Plasmids and methods for peptide display and affinity-selection on virus-like particles of rna bacteriophages

ABSTRACT

The present invention relates to a system and method for controlling peptide display valency on virus-like particles (VLPs), especially including MS2 VLPs. In this method, large amounts of wild-type and low quantities of single-chain dimer coat proteins may be produced from a single RNA. Valency is controlled in immunogen (vaccine) production by providing a system that allows the production of large amounts of wild-type and low quantities of single-chain dimer coating proteins from a single RNA, allowing facile adjustment of display valency levels on VLPs, especially MS2 VLPS over a wide range, from few than one-on average—to as many as ninety per particle. This facilitates the production of immunogens and vaccines, including VLPs exhibiting low valency. Nucleic acid constructs useful in the expression of virus-like particles are disclosed, comprised of a coat polypeptide of MS2 modified by insertion of a heterologous peptide, wherein the heterologous peptide is displayed on the virus-like particle and encapsidates MS2 niRNA. Nucleic acid constructs are also disclosed which are useful in the expression of virus-like particles comprised of a coat polypeptide of PP7 modified by insertion of a heterologous peptide, wherein the heterologous peptide is displayed on the virus-like particle and encapsidates PP7 mRNA.

RELATED APPLICATIONS AND GRANT SUPPORT

This application claims the benefit of priority from U.S. ProvisionalPatent Application 61/335,122 filed Dec. 31, 2009, entitled “Control ofPeptide Display Valency on VLPs”, U.S. Provisional Application61/335,120, filed Dec. 31, 2009, entitled “Plasmid Vectors for FacileConstruction of Random Sequence Peptide Libraries on Bacteriophage MS2VLPs and Related Constructs, Libraries, and Methods,” and U.S.Provisional Application 61/335,121, filed Dec. 31, 2009, entitled“Peptide Display on Virus-Like Particles of Bacteriophage PP7”, thecontents of each application being incorporated herein by reference intheir entirety.

This patent application was supported by Grant Nos. GM042901 and R01AI065240 from the National Institutes of Health. The U.S. Government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to a system and method for display ofpeptides on virus-like particles (VLPs) of RNA bacteriophages,especially MS2 and PP7. Methods and plasmid vectors are described thatfacilitate the construction of high complexity random sequence andantigen frament libraries, from which peptides with desired bindingfunctions may be isolated by affinity selection. Since the density ofpeptide display is an important determining factor in the stringency ofaffinity selection, a method and plasmids are also described forcontrolling peptide display valency on the VLPs. The inventive methodallows facile adjustment of display valency levels on VLPs, especiallyMS2 VLPs over a wide range (i.e., from fewer than one on average to asmany as ninety per particle), thus facilitating the identification andproduction of immunogens and vaccines, including VLPs exhibiting lowvalency. Although this system has been developed primarily with a viewto vaccine discovery, it is has utility in a variety of otherapplications, including the identification of peptide-VLPs with utilityfor cell or tissue type-specific targeted delivery of drugs and imagingagents, and in the templated synthesis of novel materials.

This application describes methods and plasmid vectors that facilitateimplementation of the VLP display system. The invention provides nucleicacid constructs useful in the expression of virus-like particlescomprised of a coat polypeptides of MS2 or of PP7, each modified byinsertion of a heterologous peptide, wherein the heterologous peptide isdisplayed on the virus-like particle and encapsidates the specific RNA(either MS2 or PP7) that directs the synthesis of the VLP and of thepeptide displayed upon it. Related virus-like particles, methods, andimmunogenic compositions are also provided.

BACKGROUND OF THE INVENTION

VLPs as vaccines. The growth of recombinant DNA technology in recentyears has led to the introduction of vaccines in which an immunogenicprotein has been identified, cloned and expressed in a suitable host toobtain sufficient quantities of protein to allow effective protectiveimmunization in both animals and humans. Many of the most effectivevaccines are based on the potent ability of virion surfaces to elicitneutralizing antibodies. These include licensed killed or attenuatedvirus vaccines, such as polio, influenza and rabies, which effectivelyinduce protective antibody responses. More recently, subunit vaccinesbased upon self-assemblages of the structural proteins of humanpapillomavirus (HPV) and hepatitis B virus (HBV) have been approved bythe Food and Drug Administration. The subunits are expressed in asuitable host and then self-assemble into particles that structurallyresemble authentic viruses, but are noninfectious because they lack theviral genome. These so-called virus-like particles (VLPs) in general arehighly immunogenic, because the structural proteins of which they arecomprised are present in multiple copies in each individual particle.This high density of antigen presentation makes these particlesespecially effective at provoking a robust antibody response. The HBVand HPV vaccines are based on VLPs assembled from the structuralproteins of the respective viruses themselves, but VLPs can also beutilized as scaffolds for the high-density display of heterologousepitopes. Since VLPs in general represent highly repetitive and,therefore, highly immunogenic structures, they may be derived from anynumber of different virus types. The present method is directed towardutilizing the VLPs of RNA bacteriophages (especially MS2 and PP7) bothfor immunogenic display and for epitope discovery by a method analogousto phage display [1, 2].

RNA Bacteriophages. The single-strand RNA bacteriophages are a group ofviruses found widely distributed in nature. Several have beencharacterized in great detail in terms of genome sequence, molecularbiology, and capsid structure and assembly. MS2 is perhaps thebest-studied member of the group and has been the focus of most of thework performed in the inventors's laboratories, although recent workalso exploits a related phage called PP7. MS2 has a 3569-nucleotidesingle-strand RNA genome that encodes only four proteins: maturase,coat, lysis and replicase. The viral particle is comprised of 180 coatpolypeptides, one molecule of maturase, and one copy of the RNA genome.Since the coat protein itself is entirely responsible for formation ofthe icosahedral shell, the MS2 VLP can be produced from plasmids as theproduct of a single gene. Thus, in comparison to the other phages usedfor peptide display, RNA VLPs are strikingly simple. The engineering ofMS2 and PP7 VLPs for peptide display and affinity selection has beenpresented recently by these inventors [1, 2] and is also described laterin this document.

Epitope identification by conventional phage display. Phage display isone of several technologies that make possible the presentation of largelibraries of random amino acid sequences with the purpose of selectingfrom them peptides with certain specific functions (e.g. the ability tobind a specific antibody). The most commonly used phage display methodis based on the filamentous phages (e.g. M13). The basic idea is tocreate recombinant bacteriophage genomes containing a library ofrandomized sequences genetically fused in the phage's DNA genome to oneof the viral structural proteins. When such recombinants are transfectedinto bacteria, each produces a virus particle that displays a particularpeptide on its surface and packages the same recombinant genome thatencodes the peptide. This establishes the linkage of genotype andphenotype essential to the method. Arbitrary functions (e.g. the bindingof a receptor, immunogenicity) can be selected from complex libraries ofpeptide-displaying phages by the use of affinity-selection followed byamplification of the selectants by growth in E. coli. In a vast libraryof peptide-displaying phages, the tiny minority able to bind aparticular receptor (e.g. a monoclonal antibody) can be affinitypurified and then amplified by propagation in E. coli. Usually severaliterative rounds of selection and amplification are sufficient to yielda relatively simple population from which individual phages displayingpeptides with the desired activity can be cloned and then characterized.When the selecting molecule is an antibody, the peptides thus identifiedrepresent epitopes recognized by the antibody, and, under appropriateconditions, may be able to evoke in an immunized patient or animal anantibody response specific for the epitope in its native antigen.

However, there are disadvantages to filamentous phage display. Mostimportantly, a quirk of filamentous phage molecular biology often makesit difficult or impossible to display peptides at the high densitiesnecessary for really potent immunogenicity. This means that althoughpeptide epitopes may be identified by affinity selection, to be usefulas immunogens (i.e. vaccines) they must usually be synthesizedchemically and then conjugated to a more immunogenic carrier.Unfortunately, the peptide frequently loses activity when thus divorcedfrom the structural context in which it resided during its affinityselection and optimization. The RNA phage VLP display system describedhere, on the other hand, creates the ability to conduct affinityselection and immungenic epitope presentation on a single platform. Thisis a consequence of combining high-density peptide epitope display withan affinity-selection capability, and means that the structuralconstraints present during the epitope's affinity selection can bemaintained during the immunization process, increasing the likelihoodthat the epitope will retain the structure necessary to elicit thedesired antibody response.

Overview of the RNA phage VLP display method. The inventors previouslydescribed a technology for peptide display and affinity selection basedon the VLPs of RNA bacteriophages, including MS2 and PP7 [1, 2], andexplain it briefly here. Development of the VLP display method requiredthat two preconditions be satisfied: First it was necessary to identifya form of the RNA phage coat protein, and a site within it thattolerated insertion of foreign peptides without disruption of itsability to properly fold and assemble into a VLP. The AB-loop on thesurface of coat protein was chosen as the site for peptide insertion.Peptides inserted here are prominently displayed on the surface of theVLP. Unfortunately, the wild-type form of coat protein is highlyintolerant of peptide insertions in the AB-loop, with the vast majority(usually >98%) leading to folding failures. Coat protein normally foldsas a dimer, ninety of which assemble into the icosahedral VLP. Theinventors engineered a novel form of coat protein to stabilize it and torender it more tolerant of AB-loop insertions. To do so they tookadvantage of the proximity of the N- and C-termini of the two identicalpolypeptide chains in the dimer (FIG. 1). By duplicating the coatprotein coding sequence and then fusing the two copies into a singlereading frame, the inventors produced a so-called single-chain dimer.This form of the protein is dramatically more stable thermodynamicallyand its folding is vastly more tolerant of peptides inserted into theAB-loop of the downstream copy of the single-chain dimer [1, 2]. Theresulting VLPs display one peptide per dimer, or ninety peptides perVLP. The second precondition for a peptide display/affinity-selectioncapability is the linkage of phenotype to genotype, as it is essentialto provide a means to amplify affinity-selected sequences. Thisrequirement was satisfied when the inventors showed that RNA phage VLPsencapsidate the messenger-RNA that directs their synthesis [1, 2]. Thismeans that the sequences of affinity-selected peptide-VLPs can beamplified by reverse transcription and polymerase chain reaction. Whenthe selection target is a monclonal antibody, the resulting affinityselected VLPs represent vaccine candidates for elicitating in animals orpatients of antibodies whose activities mimic that of the selectingantibody.

Considerations related to peptide display valency and its control. Thisapplication describes plasmid vectors that facilitate the constructionof complex random sequence and antigen fragment libraries on RNA phageVLPs and the affinity selection from such libraries of peptides thatbind specific monoclonal antibodies (or other arbitrary receptors). Notethat as originally described, the RNA phage VLP display technologypresents 90 peptides on each VLP since the peptide is inserted in oneAB-loop of a single-chain dimer, and 90 dimers make up the VLP [1, 2].The multivalency of these particles is desirable for most applicationsof the MS2 VLP. For example, the high immunogenicity of the particle isrelated to the high density of the peptides displayed, and is thus avalued property in a vaccine. However, during the affinity selectionprocess, multivalency makes it difficult to distinguish particles thatdisplay peptides with intrinsic high binding affinity for the selectiontarget from those that bind tightly only because of multiplesimultaneous weak interactions. This “avidity vs. affinity” dilemma is awell-documented complication in the selection of high affinity peptideligands using filamentous phage display [3-5]. The present inventionaddresses this issue in the VLP display system by introducing a means ofadjusting average peptide display valency levels over a wide range,i.e., from fewer than one to as many as ninety per particle. This makesit possible to alter the density of peptide display during theaffinity-selection process. Selection is conducted in several rounds,with the first round typically conducted using multivalent display, thusobtaining a relatively complex population including all peptides havingsome minimal affinity for the target. In subsequent rounds the peptidedisplay valency can be reduced, thus increasing the selectionstringency, and resulting in the isolation of peptides with higheraffinity for the antibody target, and better molecular mimics of thepreferred epitope.

OBJECTS OF THE INVENTION

It is an object of the invention to provide VLPs which display lowvalency of heterologous peptides.

It is an additional object of the invention to provide nucleic acidconstructs for producing VLPs, including VLPs which display low or highvalency of heterologous peptides.

It is still another object of the invention to provide nucleic acidconstructs for producing VLPs, including VLPS which can control thedisplay of low or high valency of heterologous peptides.

It is yet another object of the invention to provide nucleic acidconstructs for producing VLPs which can control the display of low orhigh valency of heterologous peptides for purposes of identifyingimmunogens of high affinity and using these immunogens in therapeuticand other formulations or applications.

It is an additional object of the invention to provide methods foridentifying immunogenic peptides exhibiting high affinity to a selectedantibody.

It is yet another object to incorporate high affinity peptides intoVLPs.

Is is an additional object of the invention to provide immunogenicmethods and compostions using VLPs according to the present invention.

Any one or more of these and/or other objects of the invention may bereadily gleaned from the description of the invention which follows.

SUMMARY OF THE INVENTION

The present invention relates to compositions, structures (includingplasmids), systems and methods that facilitate the construction of highcomplexity random sequence and antigen fragment peptide libraries andthat allow for controlling the valency (i.e. the density) of peptidedisplay on MS2 VLPs in order to provide a more effective means ofidentifying and providing VLPs that incorporate selectively immunogenicpeptides. The present invention represents an extension of the methods,compositions, particles, units and other disclosures which are otherwisedisclosed in US patent publication no. US2009/0054246 and applicationno. PCT/US2007/018614 (published as WO08/024427), the entire contents ofwhich are incorporated by reference herein, and which were brieflydescribed above and in [1, 2, 6, 7].

The present invention provides that selection of peptides having thehighest affinity for a given monoclonal antibody will provide the bestmolecular mimics of the native antigen, and that these peptides are themost likely to provide or induce a relevant antibody response. Thesepeptides are proposed as being particularly appropriate for inducingimmunogenicity in a patient and providing a protective response.Vaccines that are prepared from and/or incorporate these peptides aremore effective, with reduced side effects especially including vaccinesaccording to the present invention which are administered in the absenceof an adjuvant.

Plasmid vectors are described that facilitate the construction of randomsequence or antigen fragment peptide libraries on VLPs of RNA phage MS2(pDSP 1 and pDSP62) and on VLPs derived from RNA phage PP7 (pET2P7K32and pDSP7). These vectors make possible the creation of libraries havingin excess of 10¹¹ to 10¹² individual members. However, these vectorsproduce VLPs that uniformly display foreign peptides at high density(i.e. 90 per VLP). As explained above, this is a distinct advantage forvaccine applications because it confers a high level of immunogenicityto the peptide, but it often presents a problem during affinityselection because multivalency lowers the stringency of selection andmakes it hard to select preferentially the tightest binding species in apopulation.

The present invention represents a simple solution to the problem ofpeptide display valency control, a system that allows the production oflarge amounts of wild-type and low quantities of single-chain(preferably, a dimer) coat protein containing a heterologous peptide ofat least four (4) amino acids in length from a single RNA. This approachinvolves constructing plasmids like pDSP1(am), pDSP62(am), pET2P7K32(am)and pDSP7(am). These are simple variants of the plasmids describedabove, which were modified to contain a stop codon (preferably an amberstop codon), for example, in place of the codon (alanine) which normallyencodes the first amino acid of the downstream copy of the coatingprotein in the single-chain dimer. Since these plasmids have a stopcodon at the junction of the two halves of the single-chain dimer (seefor example, pDSP1), they normally produce only the unit-length,wild-type coat protein, which of course assembles into a VLP. However,in the present approach, the plasmid or a second plasmid (e.g. pNMsupA)is modified to have inserted a tRNA gene [8, 9], such as analanine-inserting suppressor tRNA gene which is expressed under controlof a promoter (e.g. lac promoter on a chloramphenicol resistant plasmidfrom a different incompatability group), such that the suppressor tRNAis produced in amounts that cause a small percentage of ribosomestranslating the coat sequence to read through the stop codon and producethe single-chain dimer, which includes the heterologous peptide. Theresulting protein, with a guest heterologous peptide preferablyinserted, for example, into its second AB-loop or at the carboxyterminus or other position within the downstream subunit, co-assembleswith wild-type protein expressed from the same mRNA to form mosaic VLPs,which exhibit low valency.

According to the present method, VLPs can be produced in a controlledfashion to present fewer than one and as many as ninety (90)heterologous peptides per VLP, preferably about one to about ten (10)heterologous peptides per VLP, more preferably about 1 to about 5heterologous peptides per VLP, more preferably about 1 to about 3heterologous peptides per VLP, and most preferably about 2 to about 4heterologous peptides per VLP. The reduction in peptide densityaccording to the present invention results in VLPs with increasedstringency of affinity-selection, allowing the ready identification ofhigh-affinity peptides, which become strongly inununogenic when laterreturned to a high display density format. Agarose gel electrophoresisand northern blots verify that the particles produced according to thepresent invention encapsidate the relevant RNA. The method of thepresent invention further provides that the valency (number of peptidesproduced per VLP) can be adjusted over a wide range by controlling theexpression level of the suppressor tRNA, for example, by adjusting thelevel of suppressor tRNA synthesis, which may be accomplishedaccordingly, for example, by expressing the tRNA from a promoter (e.g.proB or other appropriate promoter) whose activity can be modulated as afunction of inducer concentration. Valency levels can also be controlledthrough the utilization of different suppressor tRNAs, or mutantsthereof, with greater or lesser intrinsic suppression efficiencies.

One might wonder whether valency control could be achieved more simplyby co-expressing the recombinant protein together with an excess of thewild-type coat protein, thus producing mosaic capsids wherein thecontent of foreign peptides is reduced. Unfortunately, this approach isimpractical inasmuch as reducing the valency to an appropriate level(e.g. to fewer than one to a few peptides per VLP, on average) requiresthat the wild-type protein is expressed in huge excess over therecombinant peptide. Such a co-expression strategy would produce anexcess of irrelevant (i.e. non-foreign-peptide encoding) RNA, which, byits sheer abundance, would be packaged within the VLPs in preference tothe peptide-encoding RNA. Because specific encapsidation appears to beindependent of the presence of any simple packaging signal in the RNA(1), there seems to be no simple way to mark the minoritypeptide-encoding species for selective encapsidation. While it isimaginable that there are workable variants of this co-expressionapproach, these variants tend to be overly complicated. Our systemsolves this problem by producing both forms of the protein from a singlemRNA.

By way of example, the following embodiments may be used to furtherexemplify the present invention. It is noted that in each of theembodiments which are included below, MS2 coat polypeptide or PP7 coatpolypeptide may be used in the individual nucleic acid constructs andshould not be viewed as being limited to MS2 or PP7 coat polypeptideunless such limitation is appropriate within the context of thedescription.

One embodiment of the present invention provides a nucleic acidconstruct (see pDSP1, for example) comprising:

(a) a bacterial or bacteriophage promoter (e.g. as described below inthe Detailed Description of Invention section) which is operablyassociated with a coding sequence of bacteriophage MS2 single chain coatpolypeptide dimer, wherein the coat polypeptide dimer coding sequence ismodified to define a first restriction site (e.g. SalI or KpnI)positioned 5′ to that portion of the sequence which defines the coatpolypeptide dimer AB loop;

(b) a second restriction site (e.g. BamHI) positioned 3′ to the coatpolypeptide dimer coding sequence;

(c) PCR primers positioned 5′ to the first restriction site and 3′ tothe second restriction site (a definition and listing of relevant PCRprimers is given in Detailed Description of Invention).

(d) an antibiotic resistance gene (e.g. kanamycin), and (e) areplication origin for replication in a prokaryotic cell (e.g. thereplication origin from the plasmid ColE1).

In another embodiment the invention provides a nucleic acid construct(for example, see pDSP62), comprising:

(a) a bacterial or bacteriophage promoter which is operably associatedwith a coding sequence of bacteriophage MS2 single chain coatpolypeptide dimer, wherein one of the two halves of the single chaindimer sequence is modified (i.e. “codon juggled”) with multiple silentnucleotide substitutions to produce a modified dimer so that mutagenicoligonucleotide primers may be annealed specifically to said modified orunmodified half of said modified dimer (preferably, a sufficient numberof mutations within 20 nucleotide units on either side of the AB-loopthat allow the two sequences to be distinguished by hybridization of aprimer.);

(b) a first restriction site (e.g. SalI) positioned 5′ to that portionof the coat sequence that specifies the AB-loop.

(c) a second restriction site (e.g. BamHI) 3′ to the coat polypeptidedimer coding sequence, and (d) PCR primers positioned 5′ to the firstrestriction site and 3′ to the second restriction site (a definition andlisting of relevant PCR primers is given in Detailed Description ofInvention);

(e) a gene for resistance to a first antibiotic;

(f) a replication origin for replication in a prokaryotic cell;

(g) a second origin of replication from a single strand DNAbacteriophage (e.g M13 or fd); and

(h) a helper single strand DNA bacteriophage (e.g., M13, fd) modified tocontain a gene conferring resistance to a second antibiotic.

In another embodiment, relative to the nucleic acid constructs of theinvention, the coding sequence of bacteriophage MS2 (or PP7) singlechain coat polypeptide dimer further comprises a nucleic acid sequenceencoding a heterologous peptide and the construct optionally comprises atranscription terminator positioned 3′ to the second restriction site.Such nucleic acid constructs are useful in the expression of virus-likeparticles comprised of a coat polypeptide of MS2 (or PP7) modified byinsertion of a heterologous peptide, wherein the heterologous peptide isdisplayed on the virus-like particle and encapsidates MS2 (or PP7) mRNA.

In another embodiment (for example, pET2P7K32), the nucleic acidconstruct comprises:

(a) a bacterial or bacteriophage promoter which is operably associatedwith a coding sequence of bacteriophage PP7 (or MS2) single chain coatpolypeptide dimer, wherein the coat polypeptide dimer coding sequence ismodified to define a first restriction site which is located in thedownstream portion of the coat polypeptide dimer coding sequence andwhich is either positioned 5′ to, or located within, the sequence whichdefines the coat polypeptide dimer AB loop;

(b) a second restriction site positioned 3′ to the coat polypeptidedimer coding sequence;

(c) PCR primers positioned 5′ to the first restriction site and 3′ tothe second restriction site (a definition and listing of relevant PCRprimers is given in Detailed Description of Invention).

(d) an antibiotic resistance gene; and

(e) a replication origin for replication in a prokaryotic cell.

In an alternative embodiment (for example, pDSP7), the nucleic acidconstruct comprises:

(a) a bacterial or bacteriophage promoter which is operably associatedwith a coding sequence of bacteriophage PP7 single chain coatpolypeptide dimer, wherein one half of the single chain dimer sequenceis modified (i.e. “codon juggled” with multiple silent nucleotidesubstitutions so that mutagenic oligonucleotide primers may be annealedspecifically to one or the other half (preferably, a sufficient numberof mutations within 20 nucleotide units on either side of the AB-loopthat allow the two sequences to be distinguished by hybridization of aprimer);

(b) a first restriction site which is located in the downstream portionof the coat polypeptide dimer coding sequence and which is eitherpositioned 5′ to, or located within, the sequence which defines the coatpolypeptide dimer AB loop

(c) a restriction site positioned 3′ to the coat polypeptide dimercoding sequence;

(d) PCR primers 5′ positioned to the first restriction site and 3′ tothe second restriction site (a definition and listing of relevant PCRprimers is given in Detailed Description of Invention).

(e) a gene for resistance to a first antibiotic;

(f) a replication origin for replication in a prokaryotic cell;

(g) a second origin of replication from a single strand DNAbacteriophage (e.g., M13, fd);

(h) a helper single strand DNA bacteriophage (e.g., M13, fd) modified tocontain a gene conferring resistance to a second antibiotic.

In another embodiment of the present invention, a nucleic acid constructcomprises

(a) a bacterial or bacteriophage promoter which is operably associatedwith a coding sequence of bacteriophage MS2 or PP7 single chain coatpolypeptide dimer, wherein the coat polypeptide dimer coding sequence ismodified to (1) define a first restriction site which is located in thedownstream portion of the coat polypeptide dimer coding sequence andwhich is either positioned 5′ to, or located within, the sequence whichdefines the coat polypeptide dimer AB loop, and (2) to contain anucleotide sequence (NNS)_(x), where N is any nucleotide, S is aguanosine nucleotide (G) or cytidine nucleotide (C), and x is an integerfrom 1 to 500;

(b) a second restriction site positioned 3′ to the coat polypeptidedimer coding sequence;

(c) PCR primers positioned 5′ to the first restriction site and 3′ tothe second restriction site;

(d) an antibiotic resistance gene; and

(e) a replication origin for replication in a prokaryotic cell.

In certain embodiments of the present invention which are describedherein, certain of the elements which are set forth above, as providedherein, may be optionally, but preferably included within theconstructs.

In other embodiments (for example, the derivatives of the plasmidsdescribed above, called DSP1(am), pDSP62(am), pET2P7K32(am) andpDSP7(am)) control of display valency is conferred by inclusion of thefollowing additional features:

(a) a nonsense codon (for example, an amber codon as otherwise describedherein) at the junction between the upstream and downstream halves ofthe single chain dimer;

(b) a plasmid (for example, pNMsupA) that produces a suppressor tRNA(for example, an alanine-inserting amber-suppressor) able to partiallysuppress translation termination at the nonsense codon described in (a)above. This plasmid has an origin of replication from a secondincompatibility group and confers resistance to a second antibiotic,thus allowing its stable maintenance in bacteria that also contain oneof the coat protein-producing plasmids described above.

An Overview of Library Construction and Affinity Selection of VaccineCandidates. The MS2 VLP system is used here as an example, but it shouldbe understood that similar methods apply to the PP7 system alsopresented in this application.

1. Library construction. Random sequence libraries may be produced byeither of two methods. These are described in more detail later and areillustrated in FIGS. 9 b and 15. (A.) A PCR product is cloned into pDSP1between SalI and BamHI with random sequences attached in such a way asto introduce them into the AB-loop. This method is suitable forconvenient construction of relatively low complexity libraries(typically 10⁷ to 10⁸ members), but is inconvenient to scale up tohigher levels. (B.) For higher complexity libraries (e.g. 10⁹ to 10¹¹ ormore) a synthetic oligonucleotide primer is annealed to asingle-stranded version of pDSP62 and extended with DNA polymerase toproduce a double-stranded circular molecule, which is then covalentlyclosed by the action of DNA ligase (see FIG. 12). The recombinant DNAmolecules produced by either method are introduced into an appropriateexpression strain of E. coli (e.g. BL21(DE3))and where they synthesizeVLPs. The particles produced from pDSP1 or pDSP62 display foreignpeptides at a density of 90 per particle (high-density).

2. Affinity Selection is conducted, for example, by subjecting the VLPlibrary to a procedure (e.g. biopanning, or other approach to rapidlyidentify and separate peptides exhibiting high affinity for an antibody)in which a monoclonal antibody is adsorbed the surface of one or morewells of a multi-well plastic plate. The VLP library solution isincubated with the immobilized antibody, then unbound VLPs are washedaway and discarded, and any bound VLPs are eluted, usually by loweringthe solution pH. Subsequently, the eluted VLPs are thermally denaturedand the RNA they contain is copied into DNA by reverse transcriptionusing an oligonucleotide primer that anneals to the RNA near its 3′-end(well downstream of the BamHI site). The resulting cDNA is thenamplified by PCR using primers that anneal specifically upstream of thefirst restriction site (e.g. SalI) and downstream of the secondrestriction site (e.g. BamHI).

3. Recloning. The PCR product obtained above is digested with the firstand second restriction enzymes (e.g. SalI and BamHI) and then re-clonedin the appropriate expression vector. If the second selection round isto be conducted at low peptide valency, the PCR product will be clonedin pDSP1(am) or pDSP62(am) and the whole selected population will beintroduced into an E coli expression strain containing pNMsupA (see FIG.7). The VLPs thus produced will, on average, display only a few copiesof their foreign peptides (i.e. low valency), and, because they havebeen once affinity-selected, will present a much simpler population ofpeptides than was present in the initial library.

4. Additional iterative rounds of affinity-selection and re-cloning areconducted as described above. Two to four rounds or preferably, three orfour rounds (one or two at high-valency followed by one or two at lowvalency) are typically sufficient to produce a simple population of VLPsdisplaying peptides that tightly bind the selecting monoclonal antibody.When selection is deemed complete, the sequences are cloned again inpDSP1 or pDSP62, where the peptides are again displayed at high density(up to 90 peptides per capsid) for maximal immunogenicity. Individualclones are obtained and a subjected to characterization by DNA sequenceanalysis. Their affinity in vitro for the selecting antibody is assessedand their ability to elicit a desired antibody response is determined.In demonstration experiments employing well-characterized monoclonalantibody targets, the inventors have recovered peptides whose sequencesmimic those of the previously identified epitopes. Animals immunizedwith the resulting VLPs produce antibodies that recognize the epitope ofthe original antigen.

The details of the plasmids and other aspects of the invention aredescribed further in the following Detailed Description of theInvention.

Any one or more of the above embodiments and/or other embodimentsaccording to the present invention may be further readily gleaned from adescription of the invention which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the structure of the MS2 coat protein dimer. The topleft panel emphasizes the proximity of N- and C-termini of the twosubunit chains. This feature facilitated the construction of thesingle-chain dimer, which has high tolerance of foreign peptideinsertions. Also illustrated (lower left) is the high accessibility ofthe AB-loop, both in the dimer and in the intact VLP. At right is shownthe structure of the VLP itself. The coat proteins of which the VLP iscomprised adopt three slightly different conformations according to thedicates of quasiequivalence and are shown in red, blue and green. TheAB-loops are shown in yellow. Note their repetitious nature and theirexposure on the VLP surface.

FIG. 2 shows the basics of display of random sequence peptide librarieson VLPs. A library of random sequence peptide insertions is created byrecombinant DNA methods, the resulting plasmid population is introducedinto E. coli by transformation, and a library of VLPs is produced. Eachindividual VLP in the population displays a different peptide on itssurface and contains within it (in the form of mRNA) the geneticinformation for its synthesis.

FIG. 3 illustrates the process of affinity selection. A population ofVLPs representing a random sequence peptide library is incubated with amonoclonal antibody immobilized on a surface. The vast majority of VLPstypically fail to bind the antibody and are washed away and discarded.Any VLPs whose peptides exhibit binding of the antibody are thenspecifically eluted, the RNA they contain is copied into DNA by reversetranscription, amplified by PCR and then re-cloned into an expressionplasmid (e.g. pDSP62 or pDSP62(am)) for production of theaffinity-selected VLP. Selection is typically conducted iteratively(i.e. more than twice) preferably for 3 to 5 rounds.

FIG. 4 shows the importance of peptide dislay valency. Multivalentdisplay can make it difficult to distinguish intrinsically tight bindersfrom weak binders interacting simultaneously with multiple receptors.Typically a first round of affinity selection is conducted at highvalency, but in subsequent rounds valency is reduced to a low level,thus increasing the selection stringency and ensuring the isolation ofpeptides with the highest affinity for the selecting antibody.

FIG. 5 shows plasmid pDSP1 with convenient cloning sites for insertionin the AB-loop. pDSP1 expresses the coding sequence of the single-chaindimer, modified to contain for example, unique SalI and KpnI restrictionsites. This dimer facilitates simple cloning of foreign sequences intothe AB-loop. To make these sites unique, it was necessary to destroy anumber of SalI and KpnI sites in the vector and in the upstream coatsequence.

FIG. 6 shows the plasmid pDSP62. To facilitate the production ofsingle-stranded DNA, an M13 origin of replication was introduced intothe plasmid and identified as pDSP61 and pDSP62, depending on theorientation of the M13 origin. This plasmid also contains a so-called“codon juggled” coat sequence in the upstream half of the single-chaindimer. The codon-juggled half encodes the same amino acid sequence asthe downstream half, but differs from it by containing the maximumpossible number of silent substitutions, making the two halvesdistinguishable for purposes of annealing of oligonucleotides.

FIG. 7 shows an exemplary system for production of large amounts ofwild-type and low quantities of AB-loop recombinant proteins from asingle RNA. A variant of pDSP1 (pDSP1(am)) was constructed whichcontains an amber stop codon in place of the alanine codon normallyencoding the first amino acid of the downstream copy of coat protein inthe single-chain dimer. In addition, an alanine-inserting suppressortRNA gene was synthesized and cloned which is produced in amounts thatcause a small percentage of ribosomes translating the coat sequence toread through the amber (stop) codon and produce the single-chain dimer.The resulting protein (with its guest peptide) co-assembles withwild-type protein expressed from the same mRNA to form mosaic capsidsand reduced valency as otherwise described herein.

FIG. 8 is a representation of an SDS gel electrophoresis of purifiedVLPs produced by the plasmids listed below. The point of the experimentis to show the content of the single-chain “readthrough” product, asdescribed for FIG. 7 above and in the examples section of the presentapplication, in purified VLPs. The purified VLPs were produced from thefollowing plasmids (left to right):

pDSP1 produces VLPs containing only the coat protein single-chain dimer.pDSP1-Flag produces VLPs containing only a single-chain dimer with theFlag epitope inserted in its second AB-loop.

pDSP1(am) has a nonsense mutation at the junction of coat sequences inthe single-chain dimer, causing it to produce large amounts of wild-typecoat protein and, in the presence of the suppressor-tRNA (provided frompNMsupA), small quantities of single chain dimer. Since nonsensesuppression has a low efficiency in this case, only a small fraction ofribosomes reads through the stop codon to produce the single-chaindimer. The two forms of coat protein co-assemble into a mosaic, orhybrid VLP consisting mostly of wild-type coat protein, and of smallamounts of single-chain dimer (estimated at about 3% the level ofwild-type coat protein).

pDSP1(am)-Flag is just Like pDSP1(am), but with the flag epitopeinserted at the second AB-loop of the single chain dimer. It producesmostly wild-type coat protein, and a small amount of the single-chaindimer with the flag peptide inserted in its AB-loop.

The two arrows to the right of the image indicate the positions of thetwo proteins produced by suppression of the stop codons of pDSP1(am) andpDSP1(am)-Flag.

FIGS. 9 a and 9 b depict the pDSP1 plasmid (9a) and a technique (9b) forinserting a nucleic acid sequence encoding a heterologous peptide intothat plasmid.

FIG. 10 depicts the pDSP62 plasmid and schematically shows how asynthetic oligonucleotide primer can be designed for insertion ofsequences specifically into the AB-loop of the downstream copy of thesingle-chain dimer. The oligonucleotide is designed to anneal perfectlyto the coat sequences flanking the AB-loop, and inserts a foreignsequence between then.

FIG. 11 a contains the nucleic acid sequence for the pDSP1 plasmid (SEQID NO: 1) FIG. 11 b shows the nucleic acid sequence of pDSP1(am) (SEQ IDNO: 2); FIG. 11 c shows the nucleic acid sequence for the pDSP62 plasmid(SEQ ID NO: 3).and FIG. 11 d gives the nucleic acid sequence ofpDSP62(am) (SEQ ID NO: 4). Note that the pDSP1(am) and pDSP62(am)plasmid sequences differ from pDSP1 and pDSP62 only by nucleotidesubstitutions that introduce an amber codon . FIG. 11 e is the nucleicacid sequence of M13CM1, a chloramphenicol resistant derivative ofM13K07 (SEQ ID NO: 5.)

FIG. 12 depicts a method for utilizing pDSP62 and M13CM1 to producerandom peptide sequence libraries. M13CM1 is a helper virus that allowspDSP62 to replicate from its M13 origin. Therefore, infection by M13CM1of E. coli cells containing pDSP62 allows the production ofsingle-stranded circular pDSP62. When grown in a dut⁻, ung⁻bacterialstrain the DNA becomes substituted with dUTP in place of some of thedTTP normally present in DNA. The site-directed insertion ofpeptide-encoding sequences is accomplished by extension of a syntheticoligonucleotide with DNA polymerase, and the circle is closed by theaction of DNA ligase. The resulting covalently closed circular DNA isintroduced by electroporation into a dut+, ung+ strain of E. coli, wherethe dUTP substituted strand is degraded, resulting in a high frequency(typically 85-90%) incorporation of the insertions [10].

FIGS. 13 a and 13 b depict the pP7K (13 a) and p2P7K32 (13 b) plasmids.These plasmids were utilized during the development of the PP7 VLPplatform simply for testing the insertion tolerance of the PP7 coatprotein AB-loop by translational repression and VLP assembly assays (seethe Examples Section). They should not be confused with pET2P7K32 orpDSP7, which are described later and are used for construction of VLPlibraries for affinity-selection.

FIG. 14 provides the nucleotide and amino acid sequences near PP7 coatprotein AB-loop and also provides the primer sequences that were usedfor insertion of certain specific peptide insertions. A number ofspecific and random peptide sequences were inserted to test the generaltolerance of the PP7 single-chain dimer to AB-loop insertions, and todetermine the immunogenicity of specific inserted peptides.

FIG. 15 illustrates the scheme used for construction of random sequencepeptide libraries in p2P7K32 for the purpose of demonstrating theinsertion tolerance of the PP7 single-chain coat protein's AB-loop. Thismethod is similar to that depicted in FIG. 5 b for insertion into theMS2 single-chain dimer of pDSP1.

FIG. 16 provides agarose gel electrophoresis of whole cell lysates of 24clones from each of the various libraries described herein. Nearly 100%of the clones obtained were capable of translational repression,indicating the liklihood that each protein was properly folded. Tofurther verify that the proteins had correctly folded the ability ofeach to form a VLP was assessed by subjecting a. random selection ofclones to electrophoresis and western blotting as shown in this figure.The top half of each set is the ethidium bromide stained gel, and thebottom half is a western blot of a duplicate gel. The left-most lane ineach set is the p2P7K32 control. Each clone contains a differentrandomly generated peptide sequence, The fact that nearly every oneproduces a VLP demonstrates the high level of insertion tolerance of thePP7 coat protein single-chain dimer.

FIG. 17 a depicts the pETP7K plasmid, FIG. 17 b depicts the pET2P7K32plasmid. and FIG. 17 c depicts the pDSP7 plasmid. These plasmids weregenerated for the high level over-expression of PP7 coat protein. ThepDSP7 plasmid contains a single-chain dimer of PP7 coat protein in whichone half of the sequence contains a sufficient number of nucleotidesubstitutions in the vicinity of AB-loop-encoding sequences to renderthe two halves distinguishable for purposes of annealing a mutagenicoligonucleotide. In the example shown here, the entire upstream sequenceis modified to contain the maximum number of silent mutations possible.pET2P7K32 and pDSP7 should be regarded as the PP7 analogs of the MS2coat protein producers, pDSP1 and pDSP62.

FIG. 18—Electrophoresis on formaldehyde/agarose gel of RNAs extractedfrom VLPs produced in bacteria containing pETP7K or pET2P7K32 shows thatthe VLPs they produce encapsidate the mRNAs that direct their synthesis.This provides the means of recovering affinity-selected sequences byreverse transcription and PCR. Alternate lanes contain RNAs produced bytranscription in vitro of the same plasmids. The left panel shows theethidium stained gel. On the right is a blot of a duplicate gel probedwith an oligonucleotide specific for the PP7 coat protein sense-strand.This probe fails to react with similar quantities of RNA derived from invitro transcription of MS2 or Qβ coat protein sequences (not shown).

FIG. 19 provides a list of several specific peptide sequences clonedinto the PP7 coat protein.

FIG. 20 illustrates that an anti-L2 mAb (RG-1) binds to PP7 L2-VLPs, butnot PP7 V3-VLPs. Dilutions of mAb RG-1 were reacted with 500 ng/well ofL2-VLPs or V3-VLPs. Binding was detected using a horseradishperoxidase-labeled goat anti-mouse IgG secondary followed by developmentwith ABTS. Reactivity was determined by measurement of the absorbance at405 nm (OD 405).

FIG. 21 illustrates that V3 peptide displaying PP7 VLPs induce anti-V3IgG responses upon immunization. Shown are anti-V3 lgG antibodyresponses in mice immunized with PP7 V3-VLPs or, as a control, L2-VLPs.Mice were immunized three times with 10 μg of VLPs with incompleteFreund's adjuvant and then sera were collected two weeks after the finalboost. Diluted sera from seven individual mice (six immunized withV3-VLPs and one immunized with L2-VLPs) were tested for reactivity witha peptide representing a portion of the V3 loop from HIV_(LAI) by ELISA.Binding was detected using a horseradish peroxidase-labeled goatanti-mouse IgG secondary followed by development with ABTS. Reactivitywas determined by measurement of the absorbance at 405 nm (OD 405).

FIG. 22 shows the results of affinity selection using MS2 VLP displaywith the anti-Flag M2 antibody whose epitope sequence has beenthoroughly characterized previously by others. The sequences of a fewpeptides from each of the four rounds are shown to illustrate theprogress of selection. Peptides found in rounds 1 and 2 containrecognizable elements of the native epitope, but by rounds 3 and 4 thesimilarity is obvious, the sequences closely matching that of thewild-type epitope . In fact the round 4 sequence matches the nativeepitope more closely than the sequences obtained previously by thewell-established filamentous phage display method (see “NEB Transcript,Summer, 1006—available at the New England Biolabs web site,www.neb.com).

FIGS. 23 a-23 d show the nucleotide sequences of the pET2P7K32 (SEQ IDNO: 6), pET2P7K32(am) (SEQ ID NO: 7), pDSP7 (SEQ ID NO: 8), andpDSP7(am) (SEQ ID NO: 9) plasmids. These are the PP7 coatprotein-producing analogs of the corresponding MS2 VLP producers shownin FIGS. 11 a-11 d.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, conventional molecularbiology, microbiology, and recombinant DNA techniques within the skillof the art may be employed. Such techniques are explained fully in theliterature. See, e.g., Sambrook et al, 2001, “Molecular Cloning: ALaboratory Manual”; Ausubel, ed., 1994, “Current Protocols in MolecularBiology” Volumes I-III; Celis, ed., 1994, “Cell Biology: A LaboratoryHandbook” Volumes I-Ill; Coligan, ed., 1994, “Current Protocols inImmunology” Volumes I-III; Gait ed., 1984, “Oligonucleotide Synthesis”;Hames & Higgins eds., 1985, “Nucleic Acid Hybridization”; Hames &Higgins, eds., 1984,“Transcription And Translation”; Freshney, ed.,1986, “Animal Cell Culture”; IRL Press, 1986, “Immobilized Cells AndEnzymes”; Perbal, 1984, “A Practical Guide To Molecular Cloning.”

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an” and “the” include plural references unless thecontext clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set outbelow.

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length, either ribonucleotides or deoxynucleotides,and includes both double- and single-stranded DNA and RNA. Apolynucleotide may include nucleotide sequences having differentfunctions, such as coding regions, and non-coding regions such asregulatory sequences (e.g., promoters or transcriptional terminators). Apolynucleotide can be obtained directly from a natural source, or can beprepared with the aid of recombinant, enzymatic, or chemical techniques.A polynucleotide can be linear or circular in topology. A polynucleotidecan be, for example, a portion of a plasmid or bacteriophage vector,such as an expression or cloning vector, or a fragment.

Restriction endonucleases are enzymes that cleave DNA at well-definedsequences. They are used in recombinant DNA technology, for example, togenerate specific DNA fragments that are readily joined through theaction of DNA ligase to other DNA fragments generated by digestion withthe same restriction endonuclase. In this application, reference is madeto several specific restriction endonucleases, including SalI, KpnI, andBamHI whose recognition sequences are, respectively: GTCGAC, GGTACC, andGGATCC.

As used herein, the term “polypeptide” refers broadly to a polymer oftwo or more amino acids joined together by peptide bonds. The term“polypeptide” also includes molecules that contain more than onepolypeptide joined by a disulfide bond, or complexes of polypeptidesthat are joined together, covalently or noncovalently, as multimers (eg., dimers, tetramers). Thus, the terms peptide, oligopeptide, andprotein are all included within the definition of polypeptide and theseterms are used interchangeably. It should be understood that these termsdo not connote a specific length of a polymer of amino acids, nor arethey intended to imply or distinguish whether the polypeptide isproduced using recombinant techniques, chemical or enzymatic synthesis,or is naturally occurring.

The term “single-chain dimer” refers to a normally dimeric protein whosetwo subunits have been genetically (chemically, through covalent bonds)fused into a single polypeptide chain. Specifically, in the presentinvention single-chain dimer versions of both MS2 and PP7 coat proteinswere constructed. Each of these proteins is naturally a dimer ofidentical polypeptide chains. In both the MS2 and PP7 coat proteindimers the N-terminus of one subunit lies in close physical proximity tothe C-terminus of the companion subunit (see FIG. 1). Single-chain coatprotein dimers were produced using recombinant DNA methods byduplicating the DNA coding sequence of the coat proteins and then fusingthem to one another in tail to head fashion. The result is a singlepolypeptide chain in which the coat protein amino acid appears twice,with the C-terminus of the upstream copy covalenty fused to theN-terminus of the downstream copy. Normally (wild-type) the two subunitsare associated only through noncovalent interactions between the twochains. In the single-chain dimer these noncovalent interactions aremaintained, but the two subunits have additionally been covalentlytethered to one another. This greatly stabilizes the folded structure ofthe protein and confers to it its high tolerance of peptide insertionsas described above.

This application makes frequent reference to coat protein's “AB-loop”.The RNA phage coat proteins possess a conserved tertiary structure. TheMS2 and the PP7 coat proteins, for example, each possess a structureexemplified by that of MS2 coat protein shown in FIG. 1. Each of thepolypeptide chains is folded into of a number of β-strands designated byletters A through G. The β-strands A and B form a hairpin with athree-amino acid loop connecting the two strands at the top of thehairpin, where it is exposed on the surface of the VLP. As shown in thisapplication, peptides inserted into the AB-loop are exposed on thesurface of the VLP and are strongly immunogenic.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunction is retained by the polypeptide. NH₂ refers to the free aminogroup present at the amino terminus of a polypeptide. COOH refers to thefree carboxy group present at the carboxy terminus of a polypeptide.

The term “coding sequence” is defined herein as a portion of a nucleicacid sequence that directly specifies the amino acid sequence of itsprotein product. The boundaries of the coding sequence are generallydetermined by a ribosome binding (or Shine-Dalgarno) site and atranslation initiation codon (usually AUG) in prokaryotes, or by the AUGstart codon in eukaryotes located at the start of the open readingframe, usually near the 5′-end of the mRNA, and a translation terminatorsequence (one of the nonsense codons: UAG, UGA, or UAA) located at andspecifying the end of the open reading frame, usually near the 3′-end ofthe mRNA. A coding sequence can include, but is not limited to, DNA,cDNA, and recombinant nucleic acid sequences.

As briefly noted above, a “stop codon” or “termination codon” is anucleotide triplet within messenger RNA that signals a termination oftranslation. Proteins are unique sequences of amino acids, and mostcodons in messenger RNA correspond to the addition of an amino acid to agrowing protein chain—stop codons signal the termination of thisprocess, releasing the amino acid chain. In the standard genetic code,there are three stop codons: UAG (in RNA)/TAG (in DNA) (also known as an“amber” stop codon), UAA/TAA (also known as an “ochre” stop codon), andUGA/TGA (also known as an “opal” or “umber” stop codon). Severalvariations to this predominant group are known. The use of a stop codonin the present invention will normally stop or terminate proteinsynthesis. However, there are mutations in tRNAs which allow them torecognize the stop codons, causing ribosomes to read through the stopcodon, allowing synthesis of peptides encoded downstream of the stopcodon [11-13]. For example, a mutation in the tRNA which recognizes theamber stop codon allows translation to “read through” the codon andproduce full length protein, thereby recovering the normal form of theprotein and “suppressing” the stop codon. Most often, suppression ofstop codons is only partially efficient—often only a few percent ofribosomes are permitted to read though the stop codon. In someinstances, however, suppression can be much more efficient. A fewsuppressor tRNAs simply possess higher intrinsic suppressionsefficiencies. In other cases a weak suppressor can be made moreefficient by simply expressing it at higher levels. In certainembodiments of the present invention, a stop codon is incorporated intotranscriptional units in order to control the synthesis of peptidesencoded within the transcriptional unit downstream of the stop codon. Byproviding for the controlled synthesis of tRNA which recognize the stopcodon and allow synthesis of peptides downstream of the stop codon, coatprotein may be produced which comprise a heterologous peptide within apopulation of coat proteins, the majority of which do not contain aheterologous peptide. The resulting VLPs which are assembled from thismixture of heterologous peptide containing wild-type (absence ofheterologous peptide) coat proteins result in a much lower valency ofheterologous presentation.

A “heterologous” region of a recombinant cell is an identifiable segmentof nucleic acid within a larger nucleic acid molecule that is not foundin association with the larger molecule in nature. A “heterologous”peptide is a peptide which is an identifiable segment of a polypeptidethat is not found in association with the larger polypeptide in nature.

The valency of a VLP refers to the number of copies of a heterologouspeptide displayed on the particles. A virus particle which exhibits “lowvalency” of a heterologous peptide, preferably an immunogenic peptide ofat least 4 peptide units, is a particle which displays from fewer thanone to up to about ten or more heterologous peptides in the coatpolypeptide dimers which comprise said virus particle. Virus particleswhich exhibit low valency are formed from a plurality of coatpolypeptide dimers which are free of heterologous peptide (preferably,wild-type coat polypeptide) and a minority of coat polypeptide dimerswhich comprise heterologous peptide preferably within the A-B loop ofthe downstream subunit of the coat polypeptide or at the carboxyterminus of the single chain dimer coat polypeptide, thus forming amosaic VLP.

An “origin of replication”, used within context, normally refers tothose DNA sequences that participate in DNA synthesis by specifying aDNA replication initiation region. In the presence of needed factors(DNA polymerases, and the like) an origin of replication causes DNAassociated with it to be replicated. For example, the ColE1 replicationorigin (used in plasmids like pDSP1, etc.) endows many commonly usedplasmid cloning vectors with the capacity to replicate independently ofthe bacterial chromosome. Another example is the pl5A replicationorigin, which is used in the plasmid pNMsupA, described elsewhere inthis proposal (see Figure7). The presence on a plasmid of an additionalorigin of replication from phage M13 (e.g. as with pDSP62) confers theadditional ability to replicate using that origin when E. coli cells areinfected with a so-called helper phage (e.g. M13CMI described in thisapplication), which provides necessary protein factors. M13 replicatesintracellularly as double-stranded circular DNA, but also produces asingle-stranded circular form, which it packages within the phageparticle. These particles provide a convenient source of single-strandedcircular DNA for plasmids like pDSP62 and pDSP7 (described elsewhere inthis application), which is useful for library construction using themethod illustrated in FIG. 12.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence includes the minimum number of bases orelements necessary to initiate transcription at levels detectable abovebackground. Within the promoter will be found DNA sequences responsiblefor the binding of RNA polymerase and any of the associated factorsnecessary for transcription initiation. In bacteria promoters normallyconsist of −35 and −10 consensus sequences and a more or less specifictranscription initiation site. Eukaryotic promoters will often, but notalways, contain “TATA” boxes and “CAT” boxes. Bacterial expressionvectors (usually plasmids or phages) typically utilize promoters derivedfrom natural sources, including those derived from the E. coli Lactose,Arabinose, Tryptophan, and ProB operons, as well as others frombacteriophage sources. Examples include promoters from bacteriophageslambda, T7, T3 and SP6.

In bacteria, transcription normally terminates at specific transcriptiontermination sequences, which typically are categorized as rho-dependentand rho-independent (or intrinsic) terminators, depending on whetherthey require the action of the bacterial rho-factor for their activity.These terminators specify the sites at which RNA polymerase is caused tostop its transcription activity, and thus they largely define the3′-ends of the RNAs, although sometimes subsequent action ofribonucleases further trims the RNA.

An “antibiotic resistance gene” refers to a gene that encodes a proteinthat renders a bacterium resistant to a given antibiotic. For example,the kanamycin resistance gene directs the synthesis of aphosphotransferase that modifies and inactivates the drug. The presenceon plasmids (e.g. pDSP1) of a kanamycin resistance gene provides amechanism to select for the presence of the plasmid within transformedbacteria. Similarly, the chloramphenicol resistance gene allows bacteriato grow in the presence of the drug by producing an acetyltransferaseenzyme that inactivates the antibiotic through acetylation. In thepresent application chloramphenicol resistance is used to ensure themaintenance within bacteria of pNMsupA and M13CM1.

“Reverse transcription and PCR” are presented in this application as ameans of amplifying the nucleic acid sequences of affinity-selectedVLPs. “Reverse transcription” refers to the process by which a DNA copyof an RNA molecule (or cDNA) is produced by the action of the enzymereverse transcriptase. In the present application, reverse transcriptionis used to produce a DNA copy of RNA sequences encapsidated withaffinity-selected VLPs. The reverse transcriptase enzyme requires aprimer be annealed to the RNA (see below).

The term “PCR” refers to the polymerase chain reaction, a technique usedfor the amplification of specific DNA sequences in vitro. The term “PCRprimer” refers to DNA sequences (usually synthetic oligonucleotides)able to anneal to a target DNA, thus allowing a DNA polymerase (e.g. TaqDNA polymerase) to initiate DNA synthesis. Pairs of PCR primers are usedin the polymerase chain reaction to initiate DNA synthesis on each ofthe two strands of a DNA and to thus amplify the DNA segment between thetwo primers, as illustrated, for example, in FIGS. 9 b and 15.

Examples of primers used for reverse transcription and PCR are givenhere. E2: 5′ TCA GCG GTG GCA GCA GCC AA 3′-anneals near the 3′-ends ofthe RNAs encapsdiated by the VLPs described in this application; used toprime reverse transcription.

E3.2: 5′ CGG GCT TTG TTA GCA GCC GG 3′-anneals near the 3′ends of thecDNAs generated by reverse transcription described above at a site justupstream of the E2 primer site; serves as the 3′-primer in PCR reactionsto amplify affinity-selected sequences.

The E2 and E3.2 primers contain sequences common to pDSP1, pDSP1(am),pDSP62, pDSP62(am), pET2P7K32, pET2P7K32(am), pDSP7, and pDSP7(am) andare therefore useful for reverse transcription and PCR of RNAencapsidated by VLPs from each of these sources. PCR depends on pairingE3.2 with one of the plasmid-specific 5′-primers described below. Forsimplicity only the primers for pDSP1 and pDSP1(am) are shown, but itshould be understood that similar primers specific for the sequencesfound in the other VLPs are utilized as needed.

J2: 5′ ACT CCG GCC TCT ACG GCA AC 3′-a primer that anneals specificallyto sequences at the junction between the single-chain dimer sequences ofpDSP1. In a PCR reaction with E3.2, a DNA segment is amplified thatcontains the downstream half of the single-chain dimer (including thesequence of the peptide inserted in the AB-loop). Digestion of the PCRproduct with SalI and BamHI produces a fragment that can be insertedbetween SalI and BamHI of the relevant plasmid (see FIG. 9 a, forexample)

J2(amber): 5′ AC TCC GGC ATC TAC TAG AAC TIT AC 3′-This primer functionsexactly like J2 above, but is specific for the sequences generated formpDSP I (am).

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence. Transcriptional controlsequences are DNA regulatory sequences, such as promoters, enhancers,polyadenylation signals, terminators, and the like, that provide for theexpression of a coding sequence in a host cell. Translational controlsequences determine the efficiency of translation of a messenger RNA,usually by controlling the efficiency of ribosome binding andtranslation initiation. For example, as discussed elsewhere in thisapplication, the coat proteins of the RNA phages are well-knowntranslational repressors of the phage replicase. As coat proteinaccumulates to a sufficiently high concentration in the infected cell,it binds to an RNA hairpin that contains the translation initiationregion (Shine-Dalgarno and initiator AUG) of the phage's replicase gene.This prevents ribosome binding and shuts off replicase synthesis at atime in the viral life cycle where the transition from replication tovirus assembly occurs.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid, which normally replicate independently of thebacterial chromosome by virtue of the presence on the plasmid of areplication origin. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA.

It should be appreciated that also within the scope of the presentinvention are nucleic acid sequences encoding the polypeptide(s) of thepresent invention, which code for a polypeptide having the same aminoacid sequence as the sequences disclosed herein, but which degenerate tothe nucleic acids disclosed herein. By “degenerate to” is meant that adifferent three-letter codon is used to specify a particular amino acid.

As used herein, “epitope” refers to an antigenic determinant of apolypeptide. An epitope could comprise 3 amino acids in a spatialconformation which is unique to the epitope. Generally an epitopeconsists of at least 5 such amino acids, and more usually, consists ofat least 8-10 such amino acids. Methods of determining the spatialconformation of amino acids are known in the art, and include, forexample, x-ray crystallography and 2-dimensional nuclear magneticresonance.

As used herein, a “mimotope” is a peptide that mimics an authenticantigenic epitope. In some cases the amino acid sequence may show somesimilarities with the epitope of the original antigen, but in some caseslittle or no sequence similarity exists. In such cases the mimotopemimics the 3D structure of the epitope using a different amino acidsequence. Mimotopes may be identified that mimic even non-peptideepitopes, such as those of carbohydrate antigens.

As used herein, the term “coat protein(s)” refers to the protein(s) of abacteriophage or a RNA-phage capable of being incorporated within thecapsid assembly of the bacteriophage or the RNA-phage.

As used herein, a “coat polypeptide” is defined herein as a polypeptidefragment of the coat protein that possesses coat protein function andadditionally encompasses the full length coat protein as well orsingle-chain variants thereof.

As used herein, the term “immune response” refers to a humoral immuneresponse and/or cellular immune response leading to the activation orproliferation of B- and/or T-lymphocytes and/or and antigen presentingcells. In some instances, however, the immune responses may be of lowintensity and become detectable only when using at least one substancein accordance with the invention. “Immunogenic” refers to an agent usedto stimulate the immune system of a living organism, so that one or morefunctions of the immune system are increased and directed towards theimmunogenic agent. An “immunogenic polypeptide” is a polypeptide thatelicits a cellular and/or humoral immune response, whether alone orlinked to a carrier in the presence or absence of an adjuvant.Preferably, antigen-presenting cells may be activated.

As used herein, the term “self antigen” refers to proteins encoded bythe host's DNA and products generated by proteins or RNA encoded by thehost's DNA. In addition, proteins that result from a combination of twoor several self-molecules or that represent a fraction of aself-molecule and proteins that have a high homology or the twoself-molecules as defined above (>95%, preferably >97%, morepreferably >99%) may also be considered self. Examples of a self-antigenincludes but is not limited to ErbB-2, amyloid-beta, immunoglubulin E(IgE), gastrin, ghrelin, vascular endothelial growth factor (VEGF),interleukin (IL)-17, IL-23, IL-13, CCR5, CXCR4, nerve growth factor(NGF), angiotensin II, TRANCE/RANKL and MUC-1.

As used herein, the term “vaccine” refers to a formulation whichcontains the composition of the present invention and which is in a formthat is capable of being administered to an animal.

As used herein, the term “virus-like particle of a bacteriophage” refersto a virus-like particle (VLP) resembling the structure of abacteriophage, being non replicative and noninfectious, and usuallylacking one or more viral genes needed for propagation of thebacteriophage as an infectious virus. The VLPs of RNA bacteriophagestypically also lacking the gene or genes encoding the protein orproteins responsible for viral attachment to or entry into the host.

This definition also encompasses virus-like particles of bacteriophages,in which the aforementioned gene or genes are still present butinactive, and, therefore, also leading to non-replicative andnoninfectious virus-like particles of a bacteriophage.

“VLP of RNA bacteriophage coat protein” is defined as the capsidstructure formed from the self-assembly of one or more subunits of RNAbacteriophage coat protein and usually containing mRNA for the coatprotein itself, and optionally containing host RNA. For the purposes ofthis application RNA phage VLPs are usually assembled from 180 copies ofa wild-type coat protein dimer, or from 90 copies of a single-chaindimer coat protein, or as mosaic VLPs containing variable numbers ofwild-type dimer and single-chain dimer coat proteins totaling 90 perparticle.

A nucleic acid molecule is “operatively linked” to, or “operablyassociated with” an expression control sequence when the expressioncontrol sequence controls and regulates the transcription andtranslation of nucleic acid sequence. The term “operatively linked”includes having an appropriate start signal (e.g., ATG) in front of thenucleic acid sequence to be expressed and maintaining the correctreading frame to permit expression of the nucleic acid sequence underthe control of the expression control sequence and production of thedesired product encoded by the nucleic acid sequence. If a gene that onedesires to insert into a recombinant DNA molecule does not contain anappropriate start signal, such a start signal can be inserted in frontof the gene.

The term “stringent hybridization conditions” are known to those skilledin the art and can be found in Current Protocols in Molecular Biology,John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limitingexample of stringent hybridization conditions is hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2.× SSC, 0.1% SDS at 50° C., preferably at 55° C., andmore preferably at 60° C. or 65° C.

Production of Virus-Like Particles

The present invention is directed to virus-like phage particles as wellas methods for producing these particles in vivo and in vitro. Normally,these particles are produced in vivo, however, the use of theseparticles may well be applied in an in vitro setting and this approachis anticipated by the present invention. The invention makes it possibleto increase laboratory complexity and reduce the time needed foriterative selection. The methods typically include producing virions invitro and recovering the virions. As used herein, producing virions “invitro” refers to producing virions outside of a cell, for instance, in acell-free system, while producing virions “in vivo” refers to producingvirions inside a cell, for instance, an Eschericia coli or Pseudomonasaeruginosa cell.

Bacteriophages

The system envisioned here is based on the properties of single-strandRNA bacteriophages (see RNA Bacteriophages, in The Bacteriophages.Calendar, RL, ed. Oxford University Press. 2005). The known viruses ofthis group attack bacteria as diverse as E. coli, Pseudomonas andAcinetobacter. Each possesses a highly similar genome organization,replication strategy, and virion structure. In particular, thebacteriophages contain a single-stranded (+)-sense RNA genome, containmaturase, coat and replicase genes, and have small (<300 angstrom)icosahedral capsids. These include but are not limited to MS2, Qβ, R17,SP, PP7, GA, M11, MX1, f4, Cb5, Cb12r, Cb23r, 7s and f2 RNAbacteriophages.

PP7 is a single-strand RNA bacteriophage of Pseudomonas aeroginosa and adistant relative to coliphages like MS2 and Qβ. Although the PP7bacteriophage normally infects Pseudomonas aeroginosa, virus-likeparticles are readily produced when the PP7 coat protein is expressed inE. coli from plasmids such as those described in FIGS. 9 and 13. It wasdetermined that PP7 coat protein is a specific RNA-binding protein,capable of repressing the translation of sequences fused to thetranslation initiation region of PP7 replicase, with specific RNAbinding activity since it represses the translational operator of PP7,but does not repress the operators of the MS2 or Qβ phages. Conditionsfor the purification of coat protein and for the reconstitution of itsRNA binding activity from disaggregated virus-like particles have beenestablished. The dissociation constant for PP7 operator RNA in vitro wasdetermined to be about 1 nM. Using a genetic system in which coatprotein represses translation of a replicase-β-galactosidase fusionprotein, amino acid residues important for binding of PP7 RNA wereidentified. Peabody, et al., Translational repression and specific RNAbinding by the coat protein of the Pseudomonas phage PP72001, J. Biol.Chem., Jun 22; 276(25):22507-13. Epub 2001 Apr. 16.

The coat proteins of several single-strand RNA bacteriophages are knowntranslational repressors, shutting off viral replicase synthesis bybinding an RNA hairpin that contains the replicase ribosome bindingsite. X-ray structure determination of RNA phages shows that homologiesevident from comparisons of coat protein amino acid sequences arereflected in the tertiary structures. The coat protein dimer, which isboth the repressor and the basic building block of the virus particle,consists of two intertwined monomers that together form a large β-sheetsurface upon which the RNA is bound. Each of the coat proteins uses acommon structural framework to bind different RNAs, thereby presentingan opportunity to investigate the basis of specific RNA-proteinrecognition. The RNA binding properties of the coat protein of PP7, anRNA bacteriophage of Pseudomonas aeroginosa whose coat protein showsonly 13% amino acid sequence identity to that of MS2 is describedherein. Also presented are the following findings: (1) the coat proteinof PP7 is a translational repressor; (2) an RNA hairpin containing thePP7 replicase translation initiation site is specifically bound by PP7coat protein both in vivo and in vitro, indicating that this structurerepresents the translational operator; and, (3) the RNA binding siteresides on the coat protein 13-sheet. A map of this site has beenpresented. Id.

For purposes of illustration, the genome of a particularlywell-characterized member of the group is utilized, MS2, which is asingle strand of (+)-sense RNA 3569 nucleotides long, encoding only fourproteins, two of which are structural components of the virion. Theviral particle is comprised of an icosahedral capsid made of 180 copiesof coat protein and one molecule of maturase protein together with onemolecule of the RNA genome. Coat protein is also a specific RNA bindingprotein. Assembly may be initiated when coat protein associates with itsspecific recognition target, an RNA hairpin near the 5′-end of thereplicase cistron (see FIG. 1B and SEQ ID NO: 1 of US20090054246,published Feb. 26, 2009, incorporated by reference in its entiretyherein). The virus particle is then liberated into the medium when thecell bursts under the influence of the viral lysis protein. Theformation of an infectious virus requires at least three components,namely coat protein, maturase and viral genome RNA, but experiments showthat the information required for assembly of the icosahedral capsidshell is contained entirely within coat protein itself. For example,purified coat protein can form capsids in vitro in a process stimulatedby the presence of RNA (Beckett et al., 1988, J. Mol Biol 204: 939-47).Moreover, coat protein expressed in cells from a plasmid assembles intoa virus-like particle in vivo (Peabody, D. S., 1990, J Biol Chem 265:5684-5689).

Coat Polypeptide

The coat polypeptide encoded by the coding region is typically at least120, preferably, at least 125 amino acids in length, and no greater thanabout 135 amino acids in length, preferably, no greater than 130 aminoacids in length. It is expected that a coat polypeptide from essentiallyany single-stranded RNA bacteriophage can be used. Examples of coatpolypeptides include but are not limited to the MS2 coat polypeptide(see, for example SEQ ID No: 2 of US published applicationUS20090054246), R17 coat polypeptide (see, for example, GenbankAccession No. P03612), PRR1 coat polypeptide (see, for example, GenbankAccesssion No. ABH03627), fr phage coat polypeptide (see, for example,Genbank Accession No. NP_(—)039624), GA coat polypeptide (see, forexample, Genbank Accession No. P07234), Qβ coat polypeptide (see, forexample, Genbank Accession No. P03615), SP coat polypeptide (see, forexample, Genbank Accession No P09673), f4 coat polypeptide (see, forexample, Genbank accession No. M37979.1) and PP7 coat polypeptide (see,for example, Genbank Accession No. PO363 0).

Examples of PP7 coat polypeptides include but are not limited to thevarious chains of PP7 Coat Protein Dimer in Complex With RNA Hairpin(e.g. Genbank Accession Nos. 2QUXR; 2QUXO; 2QUX_L; 2QUX_I; 2QUX_F; and2QUX_C). See also Example 1 herein and Peabody, et al., RNA recognitionsite of PP7 coat protein, Nucleic Acids Research, 2002, Vol. 30, No. 194138-4144. [14, 15]

The coat polypeptides useful in the present invention also include thosehaving similarity with one or more of the coat polypeptide sequencesdisclosed above. The similarity is referred to as structural similarity.Structural similarity may be determined by aligning the residues of thetwo amino acid sequences (i.e., a candidate amino acid sequence and theamino acid sequence) to optimize the number of identical amino acidsalong the lengths of their sequences; gaps in either or both sequencesare permitted in making the alignment in order to optimize the number ofidentical amino acids, although the amino acids in each sequence mustnonetheless remain in their proper order. A candidate amino acidsequence is the amino acid sequence being compared to an amino acidsequence present in for example, SEQ ID NO: 2 of U.S. Patent PublishedApplication No. US2009/0054246. A candidate amino acid sequence can beisolated from a single stranded RNA virus, or can be produced usingrecombinant techniques, or chemically or enzymatically synthesized.Preferably, two amino acid sequences are compared using the BESTFITalgorithm in the GCG package (version 10.2, Madison Wis.), or the Blastpprogram of the BLAST 2 search algorithm, as described by Tatusova, etal. (FEMS Microbial Lett 1999, 174:247-250), and available athttp://www.ncbi.nlm.nih.gov/blast/b12seq/b12.html. Preferably, thedefault values for all BLAST 2 search parameters are used, includingmatrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gapxdropoff=50, expect=10, wordsize=3, and optionally, filter on. In thecomparison of two amino acid sequences using the BLAST search algorithm,structural similarity is referred to as “identities.”

Preferably, a coat polypeptide also includes polypeptides with an aminoacid sequence having at least 80% amino acid identity, at least 85%amino acid identity, at least 90% amino acid identity, or at least 95%amino acid identity to one or more of the amino acid sequences disclosedabove. Preferably, a coat polypeptide is active. Whether a coatpolypeptide is active can be determined by evaluating the ability of thepolypeptide to form a capsid and package a single stranded RNA molecule.Such an evaluation can be done using an in vivo or in vitro system, andsuch methods are known in the art and routine. Alternatively, apolypeptide may be considered to be structurally similar if it hassimilar three dimensional structure as the recited coat polypeptideand/or functional activity.

Heterologous peptide sequences inserted into the coat polypeptide orpolypeptide may be a random peptide sequence. In a particularembodiment, the random sequence has the sequence Xaa_(n) wherein n is atleast 4, at least 6, or at least 8 and no greater than 20, no greaterthan 18, or no greater than 16, and each Xaa is independently a randomamino acid. Alternatively, the peptide fragment may have a definedsequence and possess a known functionality (e.g., antigenicity,immunogenicity). The heterologous sequence may be present at theamino-terminal end of a coat polypeptide, at the carboxy-terminal end ofa coat polypeptide, or present elsewhere within the coat polypeptide.Preferably, the heterologous sequence is present at a location in thecoat polypeptide such that the insert sequence is expressed on the outersurface of the capsid. In a particular embodiment, and as described inthe examples hereafter, the peptide sequence may be inserted into the ABloop regions the above-mentioned coat polypeptides. Examples of suchlocations include, for instance, insertion of the insert sequence into acoat polypeptide immediately following amino acids 11-17, or amino acids13-17 of the coat polypeptide. In a most particular embodiment, theheterologous peptide is inserted at a site corresponding to amino acids11-17 or particularly 13-17 of MS-2.

In certain embodiments according to the present invention, theheterologous peptide is inserted at a site corresponding to:

(a) amino acids 11-17 or particularly 13-17 of MS-2, R17 and fr coatpolypeptides;

(b) amino acids 10-16 of GA coat polypeptide

(c) amino acids 10-17 of Qβ and SP coat polypeptides;

(d) amino acids 8-11 of PP7 coat polypeptides and

(e) amino acids 9-17 of PRRI coat polypeptides.

Alternatively, the heterologous peptide may be inserted at theN-terminus or C-terminus of the coat polypeptide.

The heterologous peptide may be selected from the group consisting of anHIV peptide, a self antigen, Flag peptide, amino acid sequences derivedfrom the minor capsid protein L2 of human Papillomavirus type 16(HPV16), the V3 loop of HIV-1 gp120, Bacillus anthracis protectiveantigen, a receptor, a ligand which binds to a cell surface receptor, apeptide with affinity for either end of a filamentous phage particlespecific peptide, a metal binding peptide or a peptide with affinity forthe surface of MS2.

The heterologous peptide includes but is not limited to a peptideselected from the group consisting of an HIV peptide, a self antigen,Flag peptide, amino acid sequences derived from the minor capsid proteinL2 of human Papillomavirus type 16 (HPV16), the V3 loop of HIV-1 gp120,Bacillus anthracis protective antigen, a receptor, a ligand which bindsto a cell surface receptor, a peptide with affinity for either end of afilamentous phage particle specific peptide, a metal binding peptide ora peptide with affinity for the surface of PP7.

In order to determine a corresponding position in a structurally similarcoat polypeptide, the amino acid sequence of this structurally similarcoat polypeptide is aligned with the sequence of the named coatpolypeptide as specified above. For example, the corresponding positionof a coat polypeptide structurally similar to MS-2 coat polypeptide isaligned with SEQ ID NO: 2 (of published US Patent Application,US2009/0054246, which is incorporated by reference herein). From thisalignment, the position in the other coat polypeptide which correspondsto a given position of SEQ ID NO: 1 (also of published US Patentapplication, US2009/0054246) can be determined.

In a particular embodiment, the coat polypeptide is a single-chain dimercontaining an upstream and downstream subunit. Each subunit contains afunctional coat polypeptide sequence. The heterologous peptide may beinserted into the upstream and/or downstream subunit at the sitesmentioned herein above, e.g., preferably, the A-B loop region of thedownstream subunit. In a particular embodiment, the coat polypeptide isa single chain dimer of an MS2 coat polypeptide which may have asequence depicted in SEQ ID NO: 12 of published US Patent application,US2009/0054246, which is incorporated by reference herein.

In a particular embodiment, the coat polypeptide is a single-chain dimercontaining an upstream and downstream subunit. Each subunit contains afunctional coat polypeptide sequence. The heterologous peptide may beinserted on the upstream and/or downstream subunit at the sitesmentioned herein above, e.g., AB loop region of downstream subunit. In aparticular embodiment, the coat polypeptide is a single chain dimer of aPP7 coat polypeptide.

Preparation of Transcription Unit

The transcription unit of the present invention comprises an expressionregulatory region, (e.g., a promoter), a sequence encoding a coatpolypeptide and transcription terminator. The RNA polynucleotide mayoptionally include a coat recognition site (also referred to a“packaging signal”, “translational operator sequence”, “coat recognitionsite”). Alternatively, the transcription unit may be free of thetranslational operator sequence. The promoter, coding region,transcription terminator, and, when present, the coat recognition site,are generally operably linked. “Operably linked” or “operably associatedwith” refers to a juxtaposition wherein the components so described arein a relationship permitting them to function in their intended manner.A regulatory sequence is “operably linked” to or “operably associatedwith”, a coding region when it is joined in such a way that expressionof the coding region is achieved under conditions compatible with theregulatory sequence. The coat recognition site, when present, may be atany location within the RNA polynucleotide provided it functions in theintended manner.

The invention is not limited by the use of any particular promoter, anda wide variety of promoters are known. The promoter used in theinvention can be a constitutive or an inducible promoter. Preferredpromoters are able to drive high levels of RNA encoded by me codingregion encoding the coat polypeptide. Examples of such promoters areknown in the art and include, for instance, the lac promoter T7, T3, andSP6 promoters.

The nucleotide sequences of the coding regions encoding coatpolypeptides described herein are readily determined. An example of theclass of nucleotide sequences encoding one of the coat polypeptidesdescribed herein is nucleotides 4080-4470 of SEQ ID N0:3 (of publishedUS Patent application, US2009/0054246, incorporated by referenceherein). These classes of nucleotide sequences are large but finite, andthe nucleotide sequence of each member of the class can be readilydetermined by one skilled in the art by reference to the standardgenetic code.

Furthermore, the coding sequence of an RNA bacteriophage single chaincoat polypeptide comprises a site for insertion of a heterologouspeptide as well as a coding sequence for the heterologous peptideitself. In a particular embodiment, the site for insertion of theheterologous peptide is a restriction enzyme site.

In a particular embodiment, the coding region encodes a single-chaindimer of the coat polypeptide. In a most particular embodiment, thecoding region encodes a modified single chain coat polypeptide dimer,where the modification comprises an insertion of a coding sequence of atleast four amino acids at the insertion site. A schematic diagram of aparticular embodiment of such a transcription unit is shown in FIG. 3 ofpublished US Patent application, US2009/0054246. The transcription unitmay contain a bacterial promoter, such as a lac promoter or it maycontain a bacteriophage promoter, such as a T7 promoter and optionally,a T7 transcription terminator.

In addition to containing a promoter and a coding region encoding afusion polypeptide, the RNA polynucleotide typically includes atranscription terminator, and optionally, a coat recognition site. Acoat recognition site is a nucleotide sequence that forms a hairpin whenpresent as RNA. This is also referred to in the art as a translationaloperator, a packaging signal, and an RNA binding site. Without intendingto be limiting, this structure is believed to act as the binding siterecognized by the translational repressor (e.g., the coat polypeptide),and initiate RNA packaging. The nucleotide sequences of coat recognitionsites are known in the art and include, for instance, nucleotides in SEQID NO: 1 (see FIG. 1B of published US Patent application,US2009/0054246). Other coat recognition sequences have beencharacterized in the single stranded RNA bacteriophages R17, GA, Qβ, SP,and PP7, and are readily available to the skilled person. Essentiallyany transcriptional terminator can be used in the RNA polynucleotide,provided it functions with the promoter. Transcriptional terminators areknown to the skilled person, readily available, and routinely used.

Synthesis

As will be described in further detail below, the VLPs of the presentinvention may be synthesized in vivo by introducing transcription unitsinto bacteria, especially if transcription units contain a bacterialpromoter. Alternatively VLPs could be produced in vitro in a coupledcell-free transcription/translation system.

Assembly of VLPs Encapsidating Heterologous Substances

During their synthesis VLPs normally associate with the messenger-RNAfrom which they are produced by translation. This is important for theaffinity-selection capability of the system described in thisapplication. However, in some other applications (e.g. targeted deliveryof drugs or imaging agents) it may be desirable to introduce othersubstances into the VLP. These VLPs may be assembled by performing an invitro VLP assembly reaction in the presence of the heterologoussubstance. Specifically, purified coat protein subunits are obtainedfrom VLPs that have been disaggregated with a denaturant (usually aceticacid). The protein subunits are mixed with the heterologous substance.In a particular embodiment, the substance has some affinity for theinterior of the VLP and is preferably negatively charged.

Another method involves attaching the heterologous substance to asynthetic RNA version of the translational operator. During an in vitroassembly reaction, the RNA will tightly bind to its recognition site andbe efficiently incorporated into the resulting VLP, carrying with it theforeign substance.

In another embodiment, the substance is passively diffused into the VLPthrough pores that naturally exist in the VLP surface. In a particularembodiment, the substance is small enough to pass through these pores(in MS2 they're about 10 angstroms diameter) and has a high affinity forthe interior of the VLP.

VLP Populations

As noted above, the invention is directed to VLP populations orlibraries. The terms “population” and “libraries” in the instantspecification are used interchangeably and are thus deemed to besynonymous. In one particular embodiment, the library may be a randomlibrary; in another embodiment, the library is an antigen fragmentlibrary, a library of fragments derived from an antigenic polypeptide.

Random Libraries (Populations)

Oligonucleotides encoding peptides may be prepared. In one particularembodiment, the triplets encoding a particular amino acid have thecomposition NNS where N is A, G, C or T and S is G or T or alternativelyNNY where N is A, G, C, or T and Y is C or T. Multiple triplets areinserted into the coat protein gene, leading the insertion ofcorresponding peptides into the protein product. In order to minimizethe presence of stop codons, peptide libraries can be constructed usingoligonucleotides synthesized from custom trinucleotide phosphoramiditemixtures (available from Glen Research, Inc.) designed to moreaccurately reflect natural amino acid compositions and completelylacking stop codons.

The insertion of such random sequences into coat protein leads to thesynthesis of a population (or library) of VLPs, each particle displayinga different peptide on its surface. Such populations may be extremelylarge, consisting of billions of individual members. It is commonlyobserved that ligands specific for practically any receptor (e.g. amonoclonal antibody) are present in such libraries, although they willusually represent a tiny fraction of the whole population. Affinityselection, followed by amplification (in the case of the presentinvention by reverse transcription and PCR of encapsidated RNA) allowsthe recovery, analysis and exploitation of such rare species.

Examples

The invention may be better understood by reference to the followingnon-limiting examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.

Production Of VLPs Exhibiting Heterologous Peptides Of Low Valency.

The present application details recent advances in development of thetechnology for peptide display on MS2 VLPs, including descriptions ofnew plasmid vectors for the facile production of random sequence peptidelibraries and for control of peptide display valency. This represents anadvance of the methodologies which are described in published USapplication, US20090054246.

The present invention is directed to developing an MS2 Virus-LikeParticle (VLP) platform with an affinity selection capability analogousto that of filamentous phage display. The present invention is, in part,based on the ability of the coat proteins of RNA bacteriophages both todisplay foreign peptides and to encapsidate the same mRNAs that serve astemplates for their synthesis. The process entails the synthesis of coatprotein-random peptide fusions from plasmids in bacterial cells whichform VLPs. The VLPs are extracted from cells and subjected to affinityselection for binding to specific antibodies. Finally, RNA is extractedfrom the selected VLPs and subjected to reverse transcription and PCR torecover and amplify the encapsidated sequences, which are then clonedand reintroduced into bacteria, where they serve as templates foranother round of synthesis, assembly and selection. The process isrepeated through as many cycles as needed and, in the end, the selectedsequences are cloned for high-level bacterial expression of the selectedVLPs.

The present method, which is being developed to facilitate vaccinediscovery, among several other uses, possesses important features thatwere not available together in a single platform. First, the presentmethod ensures high immunogenicity by displaying foreign peptides asdense repetitive arrays on the surface of a virus-like particle (VLP).This results not only in vigorous immune responses to foreign antigens,but can also overcome immune tolerance and induce antibodies againstself-antigens. Second, in a process analogous to phage display, theplatforms of the present invention permit recovery and amplification ofaffinity-selected sequences from complex random sequence libraries. Thepresent invention thus combines in a single platform the ability toidentify relevant epitopes by valency limitation and affinity-selection,and to then present those epitopes to the immune system as a vaccine.Epitopes are identified and optimized by affinity-selection against anantibody target, and then, without changing platforms, may be presenteddirectly to the immune system as a vaccine. The peptides are displayedat high density without altering the structural constraints presentduring their original selection, thus increasing the likelihood thatfaithful molecular mimics of the native epitope are isolated, that theiroptimal structures are maintained during the immunization process, andthat relevant antibody responses are induced.

Plasmid Vectors for High Complexity Random Sequence Peptide LibraryConstruction on MS2 VLPs.

The first experiments utilizing MS2 VLP random sequence peptidelibraries were carried out in simple derivatives of pET3d, an ampicillinresistent plasmid with a T7 transcription unit. This turned out to be asomewhat less than optimal system for convenient production of highcomplexity libraries, however, and the inventors have since created aseries of vectors that combine a number of optional features whichappear to be important. These include:

A. A single-chain dimer with convenient cloning sites for insertion inthe AB-loop. pDSP1 (FIG. 1) expresses the coding sequence of thesingle-chain dimer, modified to contain for example, unique SalI andKpnI restriction sites in or near the AB-loop-encoding sequences.Typically a BamHI site is included downstream of the coat sequence. Thisform of the dimer facilitates simple cloning of foreign sequences intothe AB-loop. To make these sites unique, it was necessary to destroy anumber of SalI and KpnI sites in the vector and in the upstream coatsequence and in plasmid sequences.

B. Kanamycin resistance. pDSP1 confers resistance to kanamycin. Thisgreatly facilitates library construction by permitting selection oftransformed bacteria in liquid culture against an initially highbackground of untransformed cells. The ampicillin resistance conferredby the first generation plasmid vectors was unsuited to this purpose,because rapid degradation of the antibiotic by transformed E. coliresults in loss of selection after a surprisingly short time in culture.This would have allowed the overgrowth of untransformed cells, which, ofcourse, normally represent the great majority of the population, evenwhen efficient transformation methods are utilized.

C. An M13 origin of replication. Until now the inventors haveconstructed random sequence libraries by a procedure in which a PCRprimer is utilized to generate a fragment with the randomized sequenceattached at one end. The fragment is then digested with appropriaterestriction endonucleases and cloned in pDSP 1 at the unique sitesdescribed above (typically between SalI and BamHI, see FIG. 1). Usingsuch methods the inventors have made libraries consisting of up to 10⁹individual recombinants. However, libraries of this size areinconvenient to construct using these methods; the construction of muchlarger libraries is facilitated by methods that efficiently producelarger yields of recombinant DNA than are found in a typical ligationreaction. Specifically, the inventors make use of a variation of amethod for site-directed mutagenesis (2), which has been used already byothers to produce filamentous phage libraries in the 10¹¹ complexityrange (3). The method relies on extension of a mis-matched primer on adUTP-substituted single-stranded circular template. (Substitution of dTwith dU is accomplished by growth of the template DNA in a dut⁻,ung⁻host like CJ236 or BW313.) For the purpose of creating randomsequence peptide libraries the primer contains a random DNA sequenceflanked on each side by sequences complementary to coat sequences oneither side of the AB-loop. The primer is extended with DNA polymeraseand a covalently closed double-stranded circle is produced by the actionof DNA ligase. Transformation (e.g. by electroporation) of an ung⁺strainresults in strong selection for selective propagation of the mutantstrand, and results in a high yield of recombinants bearing the insertedsequences. These insertional mutagenesis reactions can be conducted onrelatively large quantities of DNA (e.g. 20 ug), enough to readilygenerate on the order of 10¹¹ individual recombinants or more byelectroporation.

To facilitate the production of single-stranded DNA, an M13 origin ofreplication was introduced into the plasmid and called it pDSP61 andpDSP62 (FIG. 2), depending on orientation of the M13 origin. Alsoconstructed was a helper phage called M13CM1. It is a derivative ofM13KO7 that replaces kanamycin resistance with chloramphenicol, makingit possible to select for the simultaneous presence of the M13cm1 helperand pDSP6, which confers kanamycin resistance. Superinfection by an M13helper phage of a dut⁻, ung⁻strain (e.g. CJ236) containing the plasmidresults in facile production of dUTP-substituted single-stranded DNA.

D. A synthetic “codon-juggled” coat gene. The desire to use primerextension mutagenesis for library construction introduced a newcomplication and necessitated the introduction into pDSP6 of a so-called“codon-juggled” coat sequence. The peptide display method of the presentinvention relies on the ability to specifically introduce foreignpeptides into only one of the two AB-loops of the single-chain dimer. Inthe scheme described above, the mutagenic primer would normally annealto sequences in both halves of the single-chain dimer resulting indouble insertions. But simultaneous insertions in both AB-loops resultin a high frequency of protein folding failures. For this reason, thesynthesis of a codon-juggled version of coat protein was accomplished.This codon-juggled version introduces the maximum possible number ofsilent nucleotide substitutions into the upstream half of thesingle-chain dimer, and thus produces a polypeptide having the wild-typecoat protein amino acid sequence. However, the presence of numerousmutations makes the juggled sequence incapable of efficiently annealingto the mutagenic oligonucleotide. In this way insertions are targeted tothe downstream half of the single-chain dimer.

Variants of pDSP1 and of pDSP62, referred to respectively as pDSP1(am)and pDSP62(am) retain all of the features of pDSP1 and PDSP62, but aremodified by site directed mutagenesis to convert the alanine codon(which specifies the first amino acid of the downstream copy of coatprotein in the single-chain dimer) to UAG, a specific nonsense or stopcodon known as the amber codon. Suppression of this stop codon allowsfor synthesis of both wild-type and single-chain dimer coat proteinsfrom a single mRNA. This results in the ability to control the averagepeptide display valency as described in more detail below.

Readthrough of the nonsense codon normally requires the presence of asuppressor tRNA specific for particular stop codon being suppressed.Suppressor tRNAs active for all three stop codons have been describedand are well known to molecular biologists. The work described hereutilizes an amber codon and therefore requires the use of anamber-suppressor tRNA, which in this case was specifically designed toinsert alanine. However, it should be understood that suppressor tRNAsspecific for each of the other stop codons (UAA and UGA), and insertinga variety of amino acids could be similarly utilized.

To produce the suppressor tRNA in bacterial cells a plasmid calledpNMsupA was produced (see FIG. 3). It is a deriviative of pACYC18 andcontains an origin of replication derived from the p15A plasmidincompatibility group. It also provides resistance to chloramphenicol.This means that it can be stably maintained in bacterial cells alreadycontaining pDSP 1(am) or pDSP62(am) which have ColE1 origins and conferresistance to kanamycin. Plasmid pNMsupA also contains the lac promoterand polylinker regions of pUC18 into which a synthetic suppressor tRNAgene was inserted. The transcription of the tRNA gene is under controlof the lac promoter, meaning that synthesis of the suppressor tRNA iscontrolled by inducers of the lac operon (e.g. IPTG). The sequence ofthe synthetic tRNA gene is modeled on that described previously byKleina et al. (4) and is shown below. It is flanked by EcoRI and PstIsites that facilitated its cloning in pNMsupA.

GAATTCGGGGCTATAGCTCAGCTGGGAGAGCGCTTGCATCTAAAGCAAGAGGTCAGCGGTTCGATCCCGCTTAGCTCCACCACTGCAG

Altering the level of suppressor tRNA is known also to alter the levelof nonsense suppression. By thus altering the level of synthesis ofsingle-chain dimer from pDSP1(am) or pDSP62(am) it was believed possibleto control the average number of peptides displayed per VLP, thusallowing control of display valency over a wide range. Although pNMsupAuses the lac promoter, it should be clear that a variety of promoterscould be used for control of suppressor tRNA synthesis, and that some(e.g. the promoter of the propionate operon (5)) are, in fact, betterable to provide well-controlled, graded responses to differentconcentrations of their respective inducers.

III. Controlling Display Valency. It is desirable to have a means ofcontrolling the number of peptides displayed on the MS2 VLP. Themultivalency of MS2 VLP display (90 copies of the epitope per particle)should make it difficult to discriminate VLPs displaying peptides withhigh intrinsic affinity for the antibody from those that have lowintrinsic affinity, but still bind tightly by virtue of their ability toengage in multiple weak interactions (i.e. avidity vs. affinity). Thisis a well-known complication of filamentous phage display, whereselection of high affinity interactions normally requires use of a lowdisplay valency. Finding appropriate molecular mimics is greatlyincreased when selecting the highest affinity peptides. Below is adescription of a method for reducing display valency so as to improvethe selection stringency.

Controlling valency. It is assumed that selection of peptides having thehighest affinity for a given monoclonal antibody will provide the bestmolecular mimics of the native antigen, and that these are the mostlikely to induce a relevant antibody response. Ideally, the approach isto conduct the first round of selection using multivalent display, thusobtaining a relatively complex population including all peptides havingsome minimal affinity for the target. It would then be desirable toreduce the display valency in subsequent rounds so as to increase thestringency of affinity selection.

In this approach, a system that allows the production of large amountsof wild-type and low quantities of AB-loop recombinant proteins from asingle RNA is provided. A variant of pDSP1 (pDSP1(am)) was constructedwhich contains an amber stop codon in place of the alanine codonnormally encoding the first amino acid of the downstream copy of coatprotein in the single-chain dimer (see FIG. 3). pDSP1(am) thereforenormally produces only wild-type coat protein, which, of course,assembles normally into a VLP. In addition, the inventors synthesizedand cloned an alanine-inserting suppressor tRNA gene. Expressed undercontrol of the lac promoter on a chloramphenicol resistent plasmid froma different incompatibility group, the suppressor tRNA is produced inamounts that cause a small percentage of ribosomes translating the coatsequence to read through the amber (stop) codon and produce thesingle-chain dimer. The resulting protein (with its guest peptide)co-assembles with wild-type protein expressed from the same mRNA to formmosaic capsids. Purified VLPs were produced from this vector and it wasestimated that they would display about three peptides per VLP, onaverage. SDS gel electrophoresis (see FIG. 4) shows the content of the“readthrough” product in purified VLPs. These were tested to confirm theincrease in stringency of affinity selection as predicted. Agarose gelelectrophoresis and northern blots verify that the particles encapsidatethe relevant RNA. If needed, the valency can be further reduced bydecreasing the expression level of the suppressor tRNA. In fact, it ispossible to be able to control the expression of the read-throughproduct (and the display valency) over a wide range by adjusting thelevel of suppressor tRNA synthesis. This is accomplished by expressingthe tRNA from a promoter (e.g. proB) whose activity can be preciselymodulated as a function of inducer concentration.

Antigen Fragment Libraries

An alternative strategy takes advantage of the existence of a clonedantigen gene or pathogen genome to create random antigen fragmentlibraries. Several methods exist for the creation of such libraries. Oneinvolves random fragmentation of the antigen gene, for example bytreatment with DNaseI to produce fragments of an appropriate averagesize (e.g. −30 bp). These are blunt-end ligated to an appropriate sitein the gene encoding the coat polypeptide (e.g. in the AB-loop of asingle-chain coat protein dimer). The resulting library is thensubjected to affinity selection to recover VLPs displaying peptidesrecognized by an antibody. In a particular embodiment, a restrictionsite may be inserted into the AB-loop or N-terminus of the coatpolypeptide.

Synthesis

RNA phage VLPs are normally produced from plasmids in living E. colicells, which are lysed and the VLPs extracted. In the experiments so farconducted by the inventors and described elsewhere in this application,random sequence peptide libraries on MS2 and PP7 VLPs have been producedexactly in this manner. However, in a particular alternative embodiment,the populations of the present invention may be synthesized in a coupledin vitro transcription/translation system using procedures known in theart (see, for example, U.S. Pat. No. 7,008,651; Kramer et al., 1999,Cell-free Coupled Transcription-translation Systems From E. coli, InProtein Expression. A Practical Approach, Higgins and Hames (eds.),Oxford University Press). In a particular embodiment, bacteriophage T7(or a related) RNA polymerase is used to direct the high-leveltranscription of genes cloned under control of a T7 promoter in systemsoptimized to efficiently translate the large amounts of RNA thusproduced (for examples, see Kim et al., 1996, Eur J Biochem 239: 881-886; Jewett et al., 2004, Biotech and Bioeng 86: 19-26).

When VLPs are produced in vivo, the E coli cell itself provides thecompartmentalization that ensures that multiple copies of a given coatprotein-peptide recombinant assemble specifically with its mRNA. Unlessa similar form of compartmentalization is provided, it is possible thatduring synthesis in vitro from a mixture (i.e. library) of templates,particularly in the population of the present invention, differentindividual coat polypeptides, distinguished by their fusion to differentpeptides, could presumably package each other's mRNAs, thus destroyingthe genotype/phenotype linkage needed for effective phage display.Moreover, because each VLP is assembled from multiple subunits,formation of hybrid VLPs may occur. Thus, in one preferred embodiment,when preparing the populations or libraries of the present invention,one or more cycles of the transcription/translation reactions areperformed in water/oil emulsions (Tawfik et al., 1998, Nat Biotechnol16: 652-6). In this now well-established method, individual templatesare segregated into the aqueous compartments of a water/oil emulsion.Under appropriate conditions huge numbers of aqueous microdroplets canbe formed, each containing on average a single DNA template molecule andthe machinery of transcription/translation. Because they are surroundedby oil, these compartments do not communicate with one another. The coatpolypeptides synthesized in such droplets should associate specificallywith the same mRNAs which encode them, and ought to assemble into VLPsdisplaying only one peptide. After synthesis, the emulsion can be brokenand the VLPs recovered and subjected to selection. In one particularembodiment, all of the transcription/translation reactions are performedin the water/oil emulsion. In one particular embodiment, only dropletscontaining only one template per droplet (VLPs displaying only onepeptide) are isolated. In another embodiment, droplets containing mixedVLPs are isolated (plurality of templates per droplet) in one or morecycles of transcription/translation reactions and subsequently VLPsdisplaying only one peptide (one template per droplet) are isolated.

Uses of VLPs and VLP Populations

There are a number of possible uses for the VLPs and VLP populations ofthe present invention. As will be described in further detail below, theVLPs may be used as immunogenic compositions, particularly vaccines, asdrug delivery devices, as biomedical imaging agents or asself-assembling nanodevices. The VLP populations of the presentinvention may be used to select suitable vaccine candidates.

Selection of Vaccine Candidates

The VLP populations or libraries of the present invention may be used toselect vaccine candidates. The libraries may be random or antigeniclibraries. Libraries of random or alternatively antigen-derived peptidesequences are displayed on the surface of VLPs, and specific targetepitopes, or perhaps mimotopes are then isolated by affinity-selectionusing antibodies. Since the VLPs encapsidate their own mRNAs, sequencesencoding them (and their guest peptides) can be recovered by reversetranscription and PCR. Individual affinity-selected VLPs aresubsequently cloned, over-expressed and purified.

Techniques for affinity selection in phage display are well developedand are directly applicable to the VLP display system of the presentinvention. Briefly, an antibody (or antiserum) is allowed to formcomplexes with the peptides on VLPs in a random sequence or antigenfragment display library. Typically the antibodies will have beenlabeled with biotin so that the complexes can be captured by binding toa streptavidin-coated surface, magnetic beads, or other suitableimmobilizing medium. After washing, bound VLPs are eluted, and RNAs areextracted from the affinity-selected population and subjected to reversetranscription and PCR to recover the coat-encoding sequences, which arethen recloned and subjected to further rounds of expression and affinityselection until the best-binding variants are obtained. A number ofschemes for retrieval of RNA from VLPs are readily imagined. Oneattractive possibility is to simply capture biotin-mAb-VLP complexes instreptavidin coated PCR tubes, then thermally denature the VLPs andsubject their RNA contents directly to RT-PCR. Many obvious alternativesexist and adjustments may be required depending on considerations suchas the binding capacities of the various immobilizing media. Once theselected sequences are recovered by RT-PCR it is a simple matter toclone and reintroduce them into E coli, taking care at each stage topreserve the requisite library diversity, which, of course, diminisheswith each round of selection. When selection is complete, each clone canbe over-expressed to produce a VLP vaccine candidate.

To establish the efficacy of affinity selection on the MS2 VLP platformselections were conducted by the methods described above using amonoclonal antibody target whose epitope is well-characterized. The M2anti-Flag monoclonal antibody, and a library constructed in pDSP1 tocontain ten NNS triplets inserted between the codons for amino acids 13and 16 (i.e. the 13/16 insertion mode). This particular librarycontained about 10⁸ independent clones and displayed foreign peptides athigh valency. Since then, more complex libraries have been constructedusing pDSP62 (see above), but the pDSP1 library was deemed sufficientlycomplex to give a reasonable probability of encountering the Flagepitope. The first selection round was conducted against 250 ng of theantibody immobilized by adsorption to plastic wells, with an estimatedten-fold excess of VLPs over antibody molecules. After extensivewashing, bound VLPs were eluted and then subjected to reversetranscription and PCR. The PCR products were digested with SalI andBamHI and cloned in pDSP62 for production of VLPs for use in round 2. Inthis, and in all subsequent rounds, cloning of the selectants yielded atleast 5×10⁶ independent clones. The second selection was conducted underthe same conditions as round 1. Products of the second and third roundswere cloned in pDSP62(am) and the VLPs were produced in the presence ofthe amber suppressor (pNMsupA) described above, meaning that in rounds 3and 4 the peptides were displayed at low valency. In the fourth roundthe amount of antibody was reduced to 50 ng, so that VLPs were presentat about 50-fold excess compared to antibody. After the fourth round,products were cloned in pDSP62 for high valency display in anticipationof overproduction and purification of VLPs. Sequences of a fewselectants from each round are shown in FIG. 22. Sequences obtained inearly rounds show limited similarity to the known Flag epitope,DYKDDDDKL, but certain key elements are already evident, includingespecially the YK dipeptide. By round three all the sequences show theDYK element together with at least one downstream D. By round four onlyone sequence was obtained from the 7 clones subjected to sequenceanalysis. Its similarity to the sequence of the wild-type flag epitopeis obvious. The Flag epitope was previously mapped using conventionalfilamentous phage display methods and the results were reported in theNEB Transcript, Summer 1996. (The NEB Transcript is a publication of NewEngland Biolabs (availabe at their web site, www. Neb.com), a commercialsupplier of phage display libraries and affinity selection kits. Thesequence reported in FIG. 22 is, in fact, a better fit to the actualepitope than that obtained in the NEB experiments, showing that the MS2VLP display system is at least as effective as filamentous phage displayfor epitope identification by affinity-selection.

Immunogenic Compositions

As noted above, the VLPs identified by the screening procedures of thepresent invention may be used to formulate immunogenic compositions,particularly vaccines. The vaccines should be in a form that is capableof being administered to an animal. Typically, the vaccine comprises aconventional saline or buffered aqueous solution medium in which thecomposition of the present invention is suspended or dissolved. In thisform, the composition of the present invention can be used convenientlyto prevent, ameliorate, or otherwise treat a condition or disorder. Uponintroduction into a host, the vaccine is able to provoke an immuneresponse including, but not limited to, the production of antibodiesand/or cytokines and/or the activation of cytotoxic T cells, antigen.presenting cells, helper T cells, dendritic cells and/or other cellularresponses.

Optionally, the vaccine of the present invention additionally includesan adjuvant which can be present in either a minor or major proportionrelative to the compound of the present invention. The term “adjuvant”as used herein refers to non-specific stimulators of the immune responseor substances that allow generation of a depot in the host which whencombined with the vaccine of the present invention provide for an evenmore enhanced immune response. A variety of adjuvants can be used.Examples include complete and incomplete Freund's adjuvant, aluminumhydroxide and modified muramyl dipeptide.

Optionally, the vaccine of the present invention additionally includesan adjuvant which can be present in either a minor or major proportionrelative to the compound of the present invention.

Targeted Drug Delivery

Affinity selection can be used to identify peptides that bind specificcellular receptors, thus producing particles able to bind and enter(e.g. by endocytosis) a targeted cell. The MS2 VLP is a hollow spherewith an internal diameter on the order of 20 nm. In a particularembodiment, the VLP comprises the drug, e.g., a protein toxin to bedelivered and optionally a ligand that binds to cell-type specificreceptors. The internal composition of such a particle may be controlledby specifically loading it, for example, with a protein toxin likericin, or by coupling it to a synthetic translational operator mimic. Byconferring the ability to bind cell type-specific receptors to the outersurface of such particles, it is possible to target delivery of thetoxin (or other drug) to selected cell types. In a related aspect, theVLP comprising the coat polypeptide dimer may actually encapsidate aheterologous substance such as a bacterial toxin, adjuvant orimmunostimulatory nucleic acid.

Biomedical Imaging Agents

In the same way that drugs can be targeted to specific cell types, socould contrast agents for magnetic resonance imaging be delivered tospecific cells or tissues, potentially increasing enormously thediagnostic power of an MRI. In fact, MS2 particles have already beenlabeled with gadolinium to greatly increase MRI contrast (Anderson etal., 2006, Nano Letters 6(6),1160-1164). Thus, in a particularembodiment, such particles could be targeted to specific sites bydisplaying appropriate receptor-specific peptides on their surfaces. Ina related aspect, the VLP comprising the coat polypeptide dimer mayactually encapsidate the imaging agent.

Self-Assembling Nano-Devices

The VLPs of the present invention may comprise peptides with affinityfor either terminus of a filamentous phage particle that displays metalbinding proteins. A VLP with affinity for either terminus of afilamentous phage particle would create the possibility of connectingthese spheres (and whatever they contain) to the ends of filamentousphage nanowires. Alternatively, the VLPs may display metal-bindingpeptides (e.g. gold and zinc) so that arrays with unusual electrical andoptical properties may be obtained. Alternatively, VLPs with improvedability to self-assemble into these arrays may be produced by displayingpeptides with affinity for a particular surface, or that alter theself-association properties of the VLPs themselves.

Experimental Overview

Two plasmid vectors that facilitate the construction of random-sequencepeptide libraries on virus-like particles (VLPs) of bacteriophage MS2are described. The first, pDSP1, was constructed for convenient cloningof PCR-generated—or other double-stranded DNA—fragments into the AB-loopof the downstream copy of a coat protein single chain dimer. The secondis called pDSP62 and was constructed specifically for introduction ofpeptide sequences at virtually any position in the single-chain dimer(usually the AB-loop) by the site-directed mutagenesis method of Kunkelet al. [16]. The general features of the plasmids are presented below.

Example 1

pDSP1—A Plasmid Expressing a Single-Chain Dimer with Convenient CloningSites for Insertion in the AB-Loop.

The plasmid pDSP1 (see FIGS. 5 a and 7 a) contains the T7 transcriptionsignals of pET3d and the kanamycin resistance and replication origin ofpET9d. (Information regarding pET3d and pET9d may be found at the NewEngland Biolabs vector database,https://www.lablife.org/ct?f=v&a=listvecinfo). It expresses the codingsequence of the MS2 single-chain coat protein dimer (6), modified tocontain unique SalI and KpnI restriction sites. This facilitates simplecloning of foreign sequences into the AB-loop. To make these sitesunique, it was necessary to destroy other SalI and KpnI sites in thevector and in the upstream coat sequence.

The MS2 coat sequence in the vicinity of the AB-loop insertion site forpDSP1 is shown below. Note the presence of SalI and KpnI sites.

... 6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22......GlnPheValLeuValAspAsnGlyGlyThrGlyAspValThrValAlaPro......CAGTTCGTTCTCGTCGACAATGGCGGTACCGGCGACGTGACTGTCGCCCA...               SalI        KpnI

Shown below is an example of a random 7-mer library in pDSP1, with therandom sequence inserted in the so-called 13/16 mode.

...  6  7  8  9 10 11 12 13                      16 17 18 19 20 21 22......GlnPheValLeuValAspAsnGly x  x  x  x  x  x  x GlyAspValThrValAlaPro......CAGTTCGTTCTCGTCGACAATGGCNNSNNSNNSNNSNNSNNSNNSGGCGACGTGACTGTCGCCCCA...               SalI

The presence of unique restriction sites in or near the sequencesencoding the AB-loop makes is possible to simply insert foreignsequences when they are flanked with sites whose cleavage generatescompatible “sticky ends”. However, it is sometimes more convenient toattach the foreign sequence using a combination of PCR and recombinantDNA methods as shown in FIG. 5 b. For example the 5′-PCR primer shownbelow could be used with a 3′ primer, which anneals to plasmid vectorsequences downstream of a Bain HI site, to generate a fragment of thecoat protein coding sequence with the random sequence inserted betweenamino acids 13 and 16. N=A, C, G, or T and S=G or C. After digestionwith SalI and BamHI, the fragment would be inserted between SalI andBamHI of pDSP1. Shown below is an example of a 5′-primer that could beused to generate such a library:

5′-CGCGTCGACAATGGC(NNS)₇GGCGACGTGACTGTCGCCCCA-3′

With pDSP1, random-sequence peptide libraries are usually constructed bycloning into the AB-loop a PCR fragment generated using a monomeric coatprotein sequence as template (e.g. pMCT). A synthetic oligonucleotide5′-primer is designed to attach a SalI (or KpnI) site and a sequence ofrandom codons (e.g. 6-10 copies of NNY) to a site just upstream of theAB-loop. A 3′-primer anneals to sequences in the plasmid vector justdownstream of BamHI. The resulting PCR product is digested with SalI (orKpnI) and BamHI and cloned at the corresponding sites of pDSP1. Thisresults in insertion of peptides into the AB-loop, the exact site ofinsertion depending on the specific design of the 5′-primer. For mostinsertions use of the SalI site is preferred as it affords moreflexibility that KpnI in selection of the insertion site. With thesemethods it is relatively straightforward to produce peptide VLPlibraries with up to 10⁸-10⁹ individual members.

To introduce a means for control of peptide display valency a derivativeof pDSP1 (called pDSP1(am) was constructed by introduction of a nonsensecodon at the junction between the halves of the single-chain dimer. Whenexpressed in the presence of a nonsense suppressor tRNA, such as thatproduced by pNMsupA, a small amount of the single-chain dimer (with itsforeign peptide) is produced. Most of the coat protein produced frompDSP 1(am), however, is synthesized in the form of the wild-type, unitlength protein. The two forms of coat protein co-assemble into a hybridparticle that displays on average only about 3 peptides. The averagelevel of display valency can be adjusted upward or downward by alteringthe expression level of the suppressor tRNA, or by employing suppressorsexhibiting greater or lesser suppression efficiencies.

Example 2

pDSP62—A Plasmid Suitable for Library Construction using EfficientSite-Directed Mutagenesis Methods.Introduction of an M13 origin of replication.

Methods for library production like that described above for pDSP1, aredifficult to scale up, because it is inconvenient to purify DNArestriction fragments in the necessary quantities. Moreover, duringligation reactions some of the DNA is inevitably diverted into uselessside-products, reducing the yield of the desired plasmid. Theconstruction of complex libraries would be facilitated by methods thatefficiently produce larger yields of the correct recombinant DNA thanare found in a typical ligation reaction. Specifically, a variation ofan old method for site-directed mutagenesis is preferred to be used,which was already by others to produce peptide libraries on filamentousphage in the 10″ complexity range (2, 6). The method is applied tosingle-stranded circular DNAs produced from a particular kind of plasmid(also know as a phagemid) that contains an M13 origin of replication.Infection with an M13 helper phage (e.g. M13K07) of a dut⁻, ung⁻strain(e.g. BW313) containing the plasmid results in facile production ofdUTP-substituted single-stranded DNA. In the actual mutagenesisreaction, a mismatched oligonucleotide primer is annealed to thesingle-stranded DNA template and is elongated using a DNA polymerase(e.g. that of T7 phage). The DNA is ligated to produce closed circularDNA, and introduced by transformation into and ung⁺strain, where themutant strand is preferentially replicated. Previous experience in theproduction of peptide-VLP libraries indicates that typically about 90%of the transformants contain the desired peptide insertions. The primerextension mutagenesis reaction can be conducted on relatively largequantities of DNA (e.g. 20 ug), enough to readily generate on the orderof 10¹¹ individual recombinants by electroporation.

To facilitate the production of single-stranded DNA, an M13 origin ofreplication was introduced into pDSP1. To do this, the M13 origin foundin pUC119 was amplified by PCR and cloned at a unique AIwNI site inpDSP1. This plasmid, called pDSP1-IG, is the progenitor to pDSP62.Because it is only an intermediate in the construction of pDSP62 I, itssequence is not shown.

Targeting Insertions to Only One Half of the Single-Chain Dimer throughthe Use of a Synthetic “Codon-Juggled” Coat Gene.

The desire to use primer-extension mutagenesis for efficient peptidelibrary construction introduced a new complication. The present displaymethod relies on the ability to specifically introduce foreign peptidesinto only one of the two AB-loops of the single-chain dimer. Using thesingle-chain dimer sequence present in pDSP1, the mutagenic primer wouldanneal to sequences in both halves, resulting in double insertions, butit is already known that insertions in both AB-loops result in a highfrequency of protein folding failures. Moreover, even if the insertionswere tolerated, an site-directed mutagenesis that failed to target onlyone half of the single-chain dimer would result in the display of twodifferent peptides on each VLPs.

For these reasons, a “codon-juggled” version of coat protein wassynthesized and exchanged for the normal upstream half of thesingle-chain dimer. The codon-juggled sequence contains the maximumpossible number of silent nucleotide substitutions, and thus produces apolypeptide having the wild-type coat protein amino acid sequence.However, the presence of numerous mutations makes the juggled sequenceincapable of efficiently annealing to the mutagenic oligonucleotide, andtherefore the mutagenic primer is specifically directed to thedownstream AB-loop sequence. Plasmid pDSP62 is shown in FIG. 6 and itssequence is provided in FIG. 7 b.

A Chloramphenicol-Resistant M13 Helper Phage for Single-Strand pDSP62Production.

Plasmid pDSP62 confers resistance to kanamycin. The helper phages (e.g.M13KO7) usually used for production of single stranded phagemid DNA alsoconfer kanamycin resistance, and are therefore unsuitable for use withthe plasmids described here. For this reason, M13CM1 was constructed, achloramphenicol resistant derivative of M13KO7. The chloramphenicolresistance gene of pACYC184 (7) was amplified by PCR using primers thatattached recognition sequences for XhoI and SacI, and the fragment wasinserted into M13KO7 in place of its kanamycin resistance gene, takingadvantage of XhoI and SacI sites that roughly flank the kanamycinresistance determinant. In the presence of kanamycin (selects for pDSP62maintenance) and chloramphenicol (selects for helper phage), cellsproduce large quantities of single-stranded plasmid DNA after infectionwith M13 CM1. Using these single-stranded templates and the method ofKunkel et al. (2), random sequence peptide libraries have been readilyproduced that contain more than 10¹⁰ individual members for [NNS]₆,[NNS]₇, [NNS]₈ and [NNS]₁₀. Significantly higher complexities arepossible with scale-up.

To introduce a means for control of peptide display valency a derivativeof pDSP62 (called pDSP62(am) was constructed by introduction of anonsense codon at the junction between the halves of the single-chaindimer. When expressed in the presence of a nonsense suppressor tRNA,such as that produced by pNMsupA, a small amount of the single-chaindimer (with its foreign peptide) is produced. Most of the coat proteinproduced from pDSP1(am), however, is synthesized in the form of thewild-type, unit length protein. The two forms of coat proteinco-assemble into a hybrid particle that displays on average only about 3peptides. The average level of display valency can be adjusted upward ordownward by altering the expression level of the suppressor tRNA, or byemploying suppressors exhibiting greater or lesser suppressionefficiencies.

As described above, virus-like particles of bacteriophage MS2 were usedfor peptide display, and it was established that MS2 coat proteinsingle-chain dimers are highly tolerant of peptide insertions and thatthey produce correctly assembled VLPs that specifically encapsidate themRNA encoding their synthesis [2]). But MS2 is only one member of alarge family of viruses whose individual members share similar molecularbiology. The plasmids and methods described above represent refinementsto the MS2 VLP display system described previously, in which theinventors had demonstrated the insertion tolerance of the MS2 coatprotein single-chain dimer and the ability of the MS2 VLP to encapsidatethe mRNA that directs its synthesis [2]. The examples that followdocument similar results recently obtained for PP7 VLPs [1], showingspecifically that the folding and assembly of the PP7 single-chain coatprotein dimer also exhibits high tolerance to foreign peptide insertionand that the VLPs thus obtained contain, in the form of mRNA, thegenetic information for their synthesis.

Here, then, is described the engineering of VLPs of PP7, a bacteriophagephage of Pseudomonas aeruginosa, for purposes of peptide display.

PP7 VLPs offer several potential advantages and improvements over theMS2 VLP. First, the particles are dramatically more stablethermodynamically, because of the presence of stabilizing inter-subunitdisulfide bonds (8). For many practical applications, includingvaccines, increased stability is a desirable trait. Second, PP7 VLPs arenot cross-reactive immunologically with those of MS2 (9). This could beimportant in vaccine or targeted drug delivery applications where serialadministration of VLPs may be necessary. Third, it is anticipated thatthe correct folding and assembly of the PP7 VLP might be more resistantto the destabilizing effects of peptide insertion, or that it might atleast show tolerance of some peptides not tolerated in MS2 VLPs.

Example 3 Design of a PP7 Peptide Display Vector.

Two general kinds of plasmid were constructed for the synthesis of PP7coat protein in E coli (see FIGS. 9 and 13). The first expresses coatprotein from the lac promoter and is used (in combination with pRZP7 -see below) to assay for coat protein's tolerance of peptide insertionsusing translational repressor and VLP assembly assays. The secondplasmid type expresses the protein from the T7 promoter andtranscription terminator. These plasmids produce large amounts of coatprotein that assembles correctly into a VLP. They also producecoat-specific mRNA with discrete 5′- and 3′-termini for encapsidationinto VLPs.

Design of the Peptide Insertion Site.

The three-dimensional structure of the PP7 capsid shows that it iscomprised of a coat protein whose tertiary structure closely mimics thatof MS2, even though the amino acid sequences of the two proteins showonly about 12% sequence identity (10)[17]. The PP7 protein possesses anAB-loop into which peptides may be inserted following a scheme similarto the one previously described for MS2 [2]. As in the MS2 case, thisbegan by mutating the PP7 coat sequence to contain a site for therestriction endonuclease KpnI, thus facilitating insertion of foreignsequences in the plasmid called pP7K (FIG. 9 a). This modificationresulted in the amino acid substitution (El IT) shown in FIG. 10. Thissubstitution was well tolerated, since the mutant coat protein repressestranslation and assembles correctly into a VLP. Again following the MS2example, it was assumed that the folding of a single chain dimer versionof PP7 coat protein would be more resistant to AB-loop insertions thanthe conventional dimer. Its construction was described previously (8),but here it is described for the first time, for use for peptidedisplay. The single-chain dimer was modified to contain a KpnI site onlyin the downstream copy of the coding sequence, producing p2P7K32 (FIG. 9b). In this design, peptides were inserted at amino acid 11, but itshould be noted that other specific insertion sites are could be used,possibly anywhere within the AB-loop. In fact, below tests are describedof two alternative insertion modes (FIGS. 10 and 11). The first iscalled the 11/11 mode because the 11^(th) amino acid appears twice—asthr on the N-terminal side of the inserted peptide, and as the wild-typeglu11 on the C-terminal side. In the so-called 11/12 mode, the insertionis flanked by thr11 on one side and ala12 on the other.

To test the general tolerance of PP7 coat protein to AB-loop insertions,libraries of random sequence peptides inserted in the AB-loop of PP7coat protein were created using the scheme shown in FIG. 11. The randomsequences consisted of 6, 8 or 10 copies of the sequence NNY (where N isany nucleotide and Y is pyrimidine). Such libraries contain 15 of the 20possible amino acids, and are therefore capable of substantialdiversity. However, by avoiding the possibility of stop codonssubsequent analysis is greatly facilitated.

Example 4 Methods to Test Individual Members of Random-Sequence PeptideLibraries for Retention of Coat Protein Function.

Like MS2 coat protein, PP7 coat is a translational repressor. Theconstruction of pRZP7, a plasmid that fuses the PP7 translationaloperator to the E. coli lacZ gene, was previously described, placingB-galactosidase synthesis under control of the coat protein'stranslational repressor activity (11). Because it confers resistance toa different antibiotic (chloramphenicol), and because it comes from adifferent incompatibility group (i.e. it uses the p15A replicationorigin), it can easily be maintained in the same E. coli strain aseither pP7K or p2P7K32, both of which confer resistance to ampicillinand use a colE1 origin. Both of these plasmids are derived from pUC119and express coat protein at relatively low levels from the lac promoter.The expression of PP7 coat protein from pP7K or p2P7K32 repressestranslation of β-galactosidase expressed from pRZP7. This makes it easyto determine whether a given peptide insertion has interfered with theability of coat protein to correctly fold, since defective coat proteinsgive blue colonies on plates containing the B-galactosidase chromogenicsubstrate known as “xgal”, whereas a properly functioning coat proteinyields white colonies.

A more rigorous test of maintenance of function is to directly assay forthe presence of VLPs in lysates of cells expressing a peptide-coatprotein recombinant. This is accomplished by electrophoresis on agarosegel of cells lysed by sonication. Ethidium bromide staining detects theRNA-containing VLP, whose presence is then confirmed by western blotanalysis using anti-PP7 serum.

The idea then is to test the peptide insertion tolerance of PP7 coatprotein and assembly by creating random-sequence peptide libraries anddetermining the fraction of clones that retain translational repressorfunction (i.e. produce white colonies) and produce a VLP. Note that asimilar test of the peptide insertion tolerance of the MS2 coat proteinsingle-chain dimer was previously reported by these inventors [2].

Example 5

PP7 coat protein folding and assembly tolerate most random 6-mer, 8-mer,and 10-mer peptide insertions.

The (NNY)₈ libraries were constructed in pP7K and p2P7K32 and introducedinto E. coli strain CSH41F-/pRZP7 by transformation. Transformants wereplated on solid medium containing xgal. The vast majority were bona fiderecombinants, since control ligations without insert fragment gave 1000×fewer colonies. Only a small number of the pP7K recombinants weretested, but it was found that all were defective for protein folding andfailed to make VLPs. This confirms the expectation from priorexperiments with MS2 [21] that the conventional dimer is normallydestabilized by peptide insertions in the AB-loop. In the single-chaindimer of p2P7K32, however, nearly 100% of colonies were white.Twenty-four white colonies from each of the libraries were transferredto duplicate 1 ml cultures. From one culture set, crude cell lysateswere prepared for agarose gel analysis of VLPs. From the other set,plasmids were isolated and subjected to restriction enzyme digestion andgel electrophoresis, verifying that all contained an insertion of theexpected length. Plasmids from the few blue colonies were also isolated.

All turned out to contain plasmids resulting from aberrant ligationevents, and generally did not contain an intact coat sequence. Virtually100% of peptide insertions were compatible with the translationalrepressor function of coat protein. That is, they produce sufficientproperly folded coat protein to repress translation like the wild-typeprotein. This result was obtained with all the NNY libraries—6-mer,8-mer and 10-mer—independent of whether they were cloned in the 11/11 or11/12 modes.

To test directly for the presence of VLPs, crude cell lysates wereprepared from each of the duplicate cultures and subjected to agarosegel electrophoresis. Gels were stained with ethidium bromide and aduplicate was blotted nitrocellulose and probed with mouse anti-PP7serum and a horseradish peroxidase labeled second antibody. A clone isregarded as positive for VLP synthesis when it contains a band in boththe stained gel and western blot. The results are shown in FIG. 13,demonstrating that nearly all of the 6-mer clones produce a VLP at somelevel, although a few show reduced yields. This was true for both the11/11 and 11/12 insertions modes. The vast majority of 8-mer clones alsoproduce a readily identifiable VLP. However, the efficiency seems todrop somewhat as insertion length increases to 10-mers, but still aclear majority of the clones produce a VLP. Note that the mobilities ofthe individual particles are variable, consistent with the expectationthat some peptides alter the surface charge of the VLP by incorporatingcharged amino acids.

Example 6

The PP7 VLP Encapsidates Coat-Specific mRNA.

For high-level expression and RNA encapsidation tests, the PP7 coatsequences of pP7K and p2P7K32 were cloned into a plasmid containing theT7 promoter and transcription terminator, producing the plasmids calledpETP7K and pET2P7K32. The plasmids, in E. coli strain BL21(DE3),synthesized large amounts of coat protein. The resulting VLPs werepurified by chromatography on Sepharose CL4B as described previously(11, 12). RNA was purified from VLPs by phenol/chloroform extraction andsubjected to electrophoresis in duplicate agarose gels containingformaldehyde. One gel was stained with ethidium bromide and the otherwas blotted to nitrocellulose (Northern blot) where PP7 sequences weredetected using a labeled synthetic oligonucleotide specific for the coatsense-strand. RNAs produced by transcription in vitro of pETP7K andpET2P7K32 with T7 RNA polymerase were utilized as standards. Each VLPcontains a predominant species whose mobility is identical to that ofthe in vitro transcription product, and which hybridizes specificallywith the PP7 coat-specific probe. It was determined that PP7 VLPsencapsidate their mRNAs, establishing the genotype-phenotype linkagenecessary for affinity selection. Note that similar tests of MS2 coatprotein's ability to encapsidate its mRNA were reported previously bythese inventors.

Example 7

Peptides displayed on PP7 VLPs are displayed to the immune system andare immunogenic.

PP7 VLPs displaying the specific peptide sequences shown in FIGS. 10 and15 were constructed. These included the so-called Flag peptide andsequences derived from the minor capsid protein L2 of humanPapillomavirus type 16 (HPV 16), the V3 loop of HIV-1 gp120, andBacillus anthracis protective antigen. To demonstrate that theseinserted peptides were indeed displayed on the surface of VLPs, theability of a monoclonal antibody against HPV 16 L2 (called RG-1) to bindto PP7 L2-VLPs was assessed by ELISA. As shown in FIG. 16, RG-1 bound toL2-VLPs, but not to PP7 VLPs that displayed the V3 peptide. Todemonstrate the immunogenicity of the VLPs, mice were immunized withV3-VLPs by intramuscular injection as described previously [2]. As shownin FIG. 17, sera from the mice were tested by ELISA and shown to havehigh titer IgG antibodies that specifically react with a syntheticversion of the V3 peptide. Note that similar tests of the immunogenicityof peptides displayed on MS2 VLPs were previously reported by theseinventors.

Example 8

The plasmids pET2P7K32 and pDSP7 are the PP7 analogs of the MS2 coatprotein producers pDSP1 and pDSP62 described above. For control ofpeptide display valency on PP7 VLPs the pET2P7K32(am) and pDSP7(am) werealso constructed. They are analogs of pDSP I (am) and pDSP7(am),respectively:

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. Any inconsistency betweenthe material incorporated by reference and the material set for in thespecification as originally filed shall be resolved in favor of thespecification as originally filed. The foregoing detailed descriptionand examples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

Literature Cited (First Set of References)

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Literature Cited (Second Set of References)

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1-151. (canceled)
 152. A nucleic acid construct comprising: (a) abacterial or bacteriophage promoter which is operably associated with acoding sequence of a bacteriophage single chain coat polypeptidesingle-chain dimer comprising two halves wherein said coat polypeptidedimer coding sequence is modified to define a site for insertion of aforeign sequence and said coat polypeptide dimer optionally comprises anucleic acid sequence which encodes a heterologous peptide; (b) areplication origin for replication in a prokaryote; and (c) anantibiotic resistance gene; and at least one of the following: (d) atranslation stop codon at the junction between the two halves of thesingle-chain dimer; (e) a restriction site positioned 5′ to that portionof the sequence which defines the coat polypeptide; (f) modification ofone of said halves of said polypepetide dimer coding sequence withmultiple silent nucleotide substitutions so that mutagenicoligonucleotide primers may be annealed to either half of said dimer,and (g) a replication origin from a single-strand DNA bacteriophage anda helper single strand DNA bacteriophage modified to contain a geneconferring resistance to a second antibiotic.
 153. A nucleic acidconstruct according to claim 152 wherein said coat polypeptide dimerfurther comprises a nucleic acid sequence encoding a heterologouspeptide.
 154. A nucleic acid construct according to claim 152 or 153comprising a restriction site positioned 5′ to that portion of thesequence which defines the coat polypeptide dimer AB loop.
 155. Anucleic acid construct according to claim 152 wherein one of said halvesof said polypepetide dimer coding sequence is modified with multiplesilent nucleotide substitutions so that mutagenic oligonucleotideprimers may be annealed _(to) either half of said dimer.
 156. A nucleicacid construct according to claim 152 wherein said coding sequence of abacteriophage single chain coat polypeptide single-chain dimer is abacteriophage single chain coat polypeptide single-chain dimer of MS2 orPP7.
 157. A nucleic acid construct according to claim 152 comprising atranslational stop codon at the junction between the two halves of saidsingle chain dimer.
 158. A nucleic acid construct according to claim 157wherein said translational stop codon is TAG, UAG, TGA, UGA, TAA or UAA.159. A nucleic acid construct according to claim 152 comprising tworestriction sites, a first restriction site positioned 5″ to thatportion of the sequence which defines the coat polypeptide dimer AB loopand a second restriction site positioned 3′ to the coat polypeptidedimer coding sequence.
 160. A nucleic acid construct according to claim159 further comprising a PCR primer positioned 5′ to the firstrestriction site and 3′ to the second restriction site.
 161. A nucleicacid construct according to claim 159 wherein said construct furthercomprises a transcription terminator positioned 5′ to said secondrestriction site.
 162. A nucleic acid construct according to claim 152wherein said bacterial or bacteriophage promoter is a T7 or lacpromoter.
 163. A nucleic acid construct according to claim 152 whereinsaid restriction site is a SalI or KpnI restriction site.
 164. A nucleicacid construct according to claim 159 wherein said first restrictionsite is a SalI or KpnI restriction site and said second restriction siteis a BamHI restriction site.
 165. A nucleic acid construct according toclaim 152 wherein said antibiotic resistance gene is a kanamycin,ampicillin or chloramphenical resistance gene.
 166. A nucleic acidconstruct according to claim 152 wherein said origin of replication iscolE1 ori or p15A.
 167. A nucleic acid construct according to claim 152wherein said replication origin from a single-strand DNA bacteriophageis a M13 replication origin.
 168. A nucleic acid construct according toclaim 156 that produces a tRNA able to at least partially suppresstranslation termination at said translation stop codon or is inassociation with a plasmid that produces a suppressor tRNA able to atleast partially suppress translation termination at said translationstop codon.
 169. A nucleic acid construct according to claim 152 whereinsaid heterologous peptide is inserted at a site in an AB loop in adownstream half of said coat polypeptide or in a carboxy-terminus oramino-terminus of said RNA bacteriophage coat polypeptide.
 170. Thenucleic acid construct according to claim 169 wherein said heterologouspeptide is selected from the group consisting of a self antigen, areceptor, a ligand which binds to a cell surface receptor, a peptidewith affinity for either end of a filamentous phage particle specificpeptide, an HIV peptide, a Flag peptide, an amino acid sequence derivedfrom the minor capsid protein L2 of human Papillomavirus type 16(HPV16), the V3 loop of HIV-1 gp120, Bacillus anthracis protectiveantigen, a metal binding peptide or a peptide with affinity for thesurface of MS2 or PP7.
 171. The nucleic acid construct according toclaim 152 comprising: (a) a bacterial or bacteriophage promoter which isoperably associated with a coding sequence of a bacteriophage singlechain coat polypeptide single-chain dimer comprising two halves whereinsaid coat polypeptide dimer coding sequence is modified to define a sitefor insertion of a foreign sequence and said coat polypeptide dimeroptionally comprises a nucleic acid sequence which encodes aheterologous peptide; (b). a replication origin for replication in aprokaryote; (c) an antibiotic resistance gene; (g) modification of oneor said halves of said polypeptide dimer coding sequence with multiplesilent nucleotide substitutions so that mutagenic oligonucleotideprimers may be annealed to either half of said dimer; and (h) areplication origin from a single-strand DNA bacteriophage and a helpersingle strand DNA bacteriophage modified to contain a gene conferringresistance to a second antibiotic.
 172. A prokaryote which has beentransformed by the nucleic acid construct according to claim
 152. 173.The prokaryote according to claim 172 which is E. coli.
 174. Avirus-like particle expressed by a prokaryote which has been transformedby a nucleic acid construct according to claim
 152. 175. A populationcomprising virus-like particles according to claim 174 wherein eachparticle comprises a coat polypeptide of MS2 or PP7 bacteriopagemodified by insertion of a heterologous peptide and wherein theheterologous peptide is displayed on the virus-like particle andencapsidates mRNA of said bacteriophage.
 176. A method for constructinga library of virus-like particles, the method comprising: (a) providinga plurality of nucleic constructs according to claim
 152. (b) treatingthe nucleic acid constructs with a restriction enzyme; (c) insertingcoding sequences for heterologous peptides into the nucleic acidconstructs to obtain a population of transcription units; and (d)expressing the transcription units and optionally, isolating thelibrary, wherein each particle comprises a coat polypeptide of abacteriophage modified by insertion of a heterologous peptide andwherein the heterologous peptide is displayed on the virus-like particleand encapsidates bacteriophage mRNA.
 177. A method for constructing alibrary of virus-like particles, the method comprising (a) providing aplurality of nucleic acid constructs according to claim 152; (b)inserting coding sequences for heterologous peptides into saidconstructs b_(y) annealing a mutagenic oligonucleotide primer to asingle-stranded circular DNA form of said nucleic acid constructs; (c)extending the primer with DNA polymerase and ligating said primer andsaid single-stranded circular DNA to produce covalently-closed DNA toobtain a population of transcription units; (d) expressing thetranscription units, and (e) optionally isolating the library, whereineach particle comprises a coat polpeptide of a bacteriphage modified byinsertion of a heterologous peptide and wherein the heterologous peptideis displayed on the virus-like particle and encapsidates bacteriphagemRNA.
 178. A method for identifying a peptide having a property ofinterest, the method comprising: (a) providing a population of thevirus-like particles of claim 174, (b) wherein each particle comprises acoat polypeptide of a bacteriophage modified by insertion of aheterologous peptide and wherein (c) the heterologous peptide isdisplayed on the virus-like particles and encapsidates bacteriophagemRNA; and (c) assaying heterologous peptides expressed on the virus-likeparticles for the property of interest.
 179. A method for isolating animmunogenic protein, the method comprising (a) identifying animmunogenic peptide from a population of virus-like particles producedaccording to the method of claim 174; (b) amplifying the identifiedimmunogenic peptide, and optionally (c) isolating said identifiedimmunogenic peptide.
 180. An immunogenic composition comprising aneffective amount of virus-like particles according to either of claim174.
 181. A nucleic acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO:
 9. 182.-190.(canceled)